Antibody fusion protein and preparation and application thereof

Abstract

An activatable antibody fusion protein comprising an antibody moiety that specifically binds to a target, an immunoglobulin Fc moiety, a masking moiety, and a cytokine moiety. The invention also relates to a preparation method of the fusion protein and application of the fusion protein in treatment and / or prevention of tumors.

Description

Antibody fusion protein and its preparation and application Technical Field

[0001] The present invention belongs to the field of biomedicine and specifically relates to an activatable multifunctional antibody fusion protein that targets tumor-specific antigens and simultaneously has the biological effects of cytokines. The present invention also relates to the preparation and application of the antibody fusion protein. Background Art

[0002] Antibody-cytokine fusion proteins (Immunokines) are a promising class of tumor immunotherapy products. They reduce the peripheral immunotoxicity of cytokines through antibody targeting and, through fusion with antibodies, extend the half-life of cytokines and enhance the immunomodulatory effects of both cytokines and antibodies. (Xue, Hsu, Fu, & Peng, 2021) The cytokine interleukin-2 (IL-2) is essential for the survival and expansion of T cells, especially natural killer CD8+ T cells and NK cells. High-dose recombinant human IL-2 aldesleukin (trade name Proleukin) was approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic renal cell carcinoma and metastatic melanoma in 1992 and 1998, respectively. However, its short half-life and high toxicity (capillary leakage and multiple organ failure) have greatly limited its clinical application. Currently, several tumor-targeted antibodies fused with IL-2 cytokines are in clinical development, including L19-IL-2 (Darleukin), GD2-IL-2, CD20-IL-2, and EpCAM-IL-2 (Pires, Hammond, & Irvine, 2021), which are in Phase II clinical trials. Although antibody conjugation can reduce the peripheral immunotoxicity of IL-2 to a certain extent, natural or IL-2Rβγ-biased IL-2 molecules can activate peripheral lymphocytes, still leading to potential peripheral immunotoxicity.

[0003] Activatable antibody-cytokine fusion proteins retain the advantages of antibody-cytokine fusion proteins. Furthermore, a shielding peptide blocks the IL-2 receptor binding site. Metalloproteinases specifically expressed in the tumor microenvironment cleave the IL-2 receptor, releasing the active IL-2 cytokine. This simultaneously extends IL-2 half-life, targets tumors, and reduces peripheral toxicity. Hsu et al. (2021) reported that an IL-2 prodrug specifically activates CD8+ T cells in the tumor microenvironment, enhancing tumor killing while exhibiting a favorable safety profile. Other antibody-conjugated IL-2 prodrug products include Werewolf's cleavable IL-2 (US11352403, WO2019222295), XILIO's shielded IL-2 cytokine (WO2021202675), and Askgenen's cytokine prodrug (WO2019173832). The present invention designs an activatable antibody fusion protein having the structure shown in Figure 1A.

[0004] Summary of the Invention

[0005] The present invention provides an activatable antibody fusion protein, exemplified by the CLDN18.2 activatable antibody fusion protein. This protein primarily consists of an anti-CLDN18.2 antibody and an IL-2 / IL-2Rα complex, coupled to IL-2 and IL-2Rα via a cleavable linker peptide. Furthermore, the Fc-coupled sugar chain is modified through genetic and cell engineering methods, further enhancing the antibody-dependent cellular activity of the fusion protein. This article describes CLDN18.2-activatable antibody fusion proteins in the form of multifunctional fusion proteins H7E12-2-Pro-IL2 (H7E12-2 antibody, a multifunctional fusion protein of an IL-12 / IL-2Rα conjugate plus an MMP14-cleavable peptide chain), 432-Pro-IL2 (432 antibody, a multifunctional fusion protein of an IL-12 / IL-2Rα conjugate plus an MMP14-cleavable peptide chain), 362-Pro-IL2 (362 antibody, a multifunctional fusion protein of an IL-12 / IL-2Rα conjugate plus an MMP14-cleavable peptide chain), and Hit2.2-Pro-IL2 (Hit2.2 antibody). , IL-12 / IL-2Rα conjugate plus a multifunctional fusion protein of an MMP14-cleavable peptide chain) as an example, using this architectural design in combination with a CLDN18.2 target antibody, the present invention discloses a multifunctional fusion protein that targets Claudin18.2, has optimized Fc function, and has the biological effect of an IL-2 / IL-2Rα complex, obtained by genetic engineering technology. The present invention also discloses the amino acid sequence encoding the multifunctional fusion protein, the architectural design, the recombinant cell containing the recombinant vector, the recombinant cell lacking the fucose modification function containing the recombinant vector, the preparation method of the multifunctional fusion protein, and the medical use of the multifunctional fusion protein.

[0006] Specifically, the technical solutions adopted by the present invention are as follows:

[0007] In a first aspect, the present invention provides an activatable antibody fusion protein, characterized in that it comprises an antibody portion that specifically binds to a target, an immunoglobulin Fc portion, a shielding portion and a cytokine portion, wherein the shielding portion is fused to the immunoglobulin Fc portion via a connecting peptide L1, and the cytokine portion is fused to the shielding portion via a cleavable connecting peptide L2.

[0008] The activatable antibody fusion protein increases the targeting of the cytokine by combining the cytokine with the antibody targeting portion / Fc portion, and prolongs the half-life of the cytokine by fusion with the Fc portion. At the same time, the shielding portion fuses with the cytokine through a cleavable linker and inhibits the activity of the cytokine, which reduces the activity of the cytokine that is detrimental to normal tissues. On the other hand, when the cytokine reaches the tumor, the protease specifically expressed in the tumor tissue cuts the cleavable linker and releases the cytokine from the shielding portion into the tumor microenvironment, as shown in the schematic diagram in Figure 1A.

[0009] In some embodiments, the activatable antibody fusion protein comprises, from the N-terminus to the C-terminus, an antibody portion that specifically binds to a target, an immunoglobulin Fc portion, a connecting peptide L1 connecting the Fc fragment and the immunoglobulin Fc portion, a shielding portion connected to the connecting peptide L1, a cleavable connecting peptide L2 connected to a cytokine, and the cytokine, as shown in the schematic diagram in Figure 1B.

[0010] In some embodiments, the target is a tumor-specific antigen, wherein the tumor-specific antigen is selected from one or more of the following groups: Claudin18.2, CA125, AFP, CEA, EGFR, HER2, B7H3, B7H6, MUC1, MUC16, GPC3, CD24, CD20. Preferably, the tumor-specific antigen is CLDN18.2, HER2, or CD20. More preferably, the tumor-specific antigen is CLDN18.2.

[0011] In some embodiments, the cytokine is selected from one or more of the group consisting of interleukin-2 (IL-2), interferon alpha (IFNα), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin-7 (IL-7), interleukin-12 (IL-12), and interleukin-21 (IL-21). Preferably, the cytokine is IL-2.

[0012] In some embodiments, the cytokine is an IL-2 wild-type polypeptide, a mutant, or a truncate, preferably an IL-2 wild-type polypeptide, an IL-2 truncate, or an IL-2 mutant. In some embodiments, the amino acid sequence of the cytokine is shown in SEQ ID NO: 27, SEQ ID NO: 74, or SEQ ID NO: 86.

[0013] In some embodiments, the shielding moiety is a receptor of the cytokine or a binding fragment thereof, or an antibody or a binding fragment thereof that specifically binds to the cytokine, which can inhibit the activity of the cytokine by binding to the cytokine.

[0014] In some embodiments, the shielding moiety inhibits the binding of IL-2 cytokine to IL-2Rαβγ and / or IL-2Rβγ on immune cells, thereby inhibiting the activity of the cytokine.

[0015] In some embodiments, the shielding moiety is selected from the group consisting of IL-2Rα, IL-2Rβ, IL-2Rγ, or mutants or truncations thereof. Preferably, the shielding moiety is IL-2Rα. In some embodiments, the amino acid sequence of the shielding moiety is as shown in SEQ ID NO:29, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:85.

[0016] In some embodiments, the portion of the antibody that specifically binds to a target is selected from the group consisting of Fab, Fab', F(ab')2, Fv, dsFv, diabody, Fd, and Fd' fragments.

[0017] In some embodiments, the antibody portion that specifically binds to the target forms an antibody structure with the immunoglobulin Fc portion comprising a heavy chain and a light chain, wherein: the amino acid sequence of the light chain is selected from the amino acid sequences shown in SEQ ID NOs: 3, 7, 11 and 15; and / or the amino acid sequence of the heavy chain is selected from the amino acid sequences shown in SEQ ID NOs: 31, 37, 35, 33 and 9, 13, 17, 19, 21, 49, 51, 53, 55, 57, 59, 61, 63, 69, 75, 77, 79.

[0018] In some embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:5; or the amino acid sequence of the light chain is as shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:31; or the amino acid sequence of the light chain is as shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9; or the amino acid sequence of the light chain is as shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:21; or the amino acid sequence of the light chain is as shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:37; or the amino acid sequence of the light chain is as shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:13; or the amino acid sequence of the light chain is as shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:35; or the amino acid sequence of the light chain is as shown in SEQ ID NO:15, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: NO:17; or the light chain amino acid sequence is as shown in SEQ ID NO:15, and the heavy chain amino acid sequence is as shown in SEQ ID NO:19; or the light chain amino acid sequence is as shown in SEQ ID NO:15, and the heavy chain amino acid sequence is as shown in SEQ ID NO:33; or the light chain amino acid sequence is as shown in SEQ ID NO:7, and the heavy chain amino acid sequence is as shown in SEQ ID NO:49; or the light chain amino acid sequence is as shown in SEQ ID NO:7, and the heavy chain amino acid sequence is as shown in SEQ ID NO:51; or the light chain amino acid sequence is as shown in SEQ ID NO:7, and the heavy chain amino acid sequence is as shown in SEQ ID NO:53; or the light chain amino acid sequence is as shown in SEQ ID NO:7, and the heavy chain amino acid sequence is as shown in SEQ ID NO:55; or the light chain amino acid sequence is as shown in SEQ ID NO:7, and the heavy chain amino acid sequence is as shown in SEQ ID NO:57; or the light chain amino acid sequence is as shown in SEQ ID NO:7, and the heavy chain amino acid sequence is as shown in SEQ ID NO: NO:59; or the light chain amino acid sequence is shown in SEQ ID NO:7; the heavy chain amino acid sequence is shown in SEQ ID NO:61; or the light chain amino acid sequence is shown in SEQ ID NO:7; the heavy chain amino acid sequence is shown in SEQ ID NO:63; or the light chain amino acid sequence is shown in SEQ ID NO:7; the heavy chain amino acid sequence is shown in SEQ ID NO:69;or the amino acid sequence of the light chain is as shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 75; or the amino acid sequence of the light chain is as shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 77; or the amino acid sequence of the light chain is as shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 79.

[0019] In some embodiments, the immunoglobulin Fc portion is selected from the constant region amino acid sequence of IgG1, IgG2, IgG3, and IgG4. Preferably, the immunoglobulin Fc portion is selected from the constant region amino acid sequence of IgG1, and its amino acid sequence is shown in SEQ ID NO: 39.

[0020] In some embodiments, the immunoglobulin Fc portion comprises one or more amino acid substitutions selected from the group consisting of S239D, S298A, I332E, and A330L, preferably S239D and I332E or S239D, I332E, and A330L, the amino acid numbering being according to the EU system.

[0021] In some embodiments, the activatable antibody fusion protein of the present invention is afucosylated. In some embodiments, the activatable antibody fusion protein of the present invention lacks fucosylation at Asn 297 in the Fc portion of the immunoglobulin, such as lacking G0F, G1F, G2F, G0F-GN, etc.

[0022] In some embodiments, the connecting peptide L1 is selected from a flexible connecting peptide comprising glycine (G) and serine (S) residues, preferably comprising (GGGGS) n Repeat, wherein n is selected from an integer of 1-6, more preferably the amino acid sequence is as shown in SEQ ID NO: 23.

[0023] In some embodiments, the cleavable linker peptide L2 is cleaved by a tumor-associated protease, thereby releasing the active cytokine.

[0024] In some embodiments, the protease is selected from matrix metallopeptidase-1 (MMP1), MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP19, MMP20, MMP21, uPA, FAPa, or cathepsin B.

[0025] For example, sequences known to be specifically cleaved by MMP14 include, for example, IFARS-L, LARA-LK, LGPSH-Y, LPPLG-F, LQIGH-L, NSPMS-L, PKLAA-I, PTPRS-Y, RKLAF-L, RPLN-LS, RRPVA-Y, SSPLN-Y, SVPSA-I, SYPRA-Y, TMLLA-L, VGPAF-L, RPRS-LL, KIPSA-L, AHPSA-L, SPRN-LR, YGPRA- I. YPAG-LR, KAPAH-L, SQPMA-Y, HTVRG-L, LKVMN-Y, NPLG-IR, PRS-LKS, SSPLA-L, RLPYP-L, FIPFP-F, TPYG-LV, IGAL A-L, RPRG-LT, IGPQF-L, VVPNN-L, SAVG-LR, VRH-LIN, PAA-LLG, PLG-IRY, HRLLS-L, YPFGS-L, PTFAH-L, ARLGY-L, FVV RA-L, GFPLM-L, PRP-LLA, VIRF-LR, PYPVP-F, HVRH-LL, RTAHN-L, AHG-ILS, DLPAG-L, SPYG-LL, VFPMS-L, RLPWS-L, R IPRF-L, PRVHH-L, PRA-LKG, SPAS-LR, SFPNP-L, SLVRF-L, VRPRP-F, RTPIG-I, AAHG-IF, YYPRA-L, TRIAY-L, VIPRP-L, RVPYG-L, PHG-FFQ, AHG-LLL, PRVEA-L, TSPVA-L, PLG-LSG, RFPRP-I, SEPFG-L, RIPAS-L, TALP-LR, GLPMH-L, VKAYN-L , QRMAS-L, KSPLG-L, RFALN-L, SIAFA-L, LPYA-LY, PRP-LYH, IPAFN-L, EVRG-LR, RYAQP-L, TPSA-LT, RGPYH-L, IPLLN- L, YPLH-LQ, VRVLH-L, PLG-ITL, LLYAS-L, RTPVG-L, TMAHP-L, and SMPRM-L.

[0026] In some embodiments, the protease is selected from caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase-cleaved caspase 11, and caspase 12.

[0027] In some embodiments, the cleavable linker peptide L2 is cleaved by matrix metallopeptidase 14. In some embodiments, the amino acid sequence of the linker peptide L2 is shown in SEQ ID NO: 25, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

[0028] In one embodiment, the fusion protein comprises, from N-terminus to C-terminus, an antibody portion that specifically binds to a target, an immunoglobulin Fc portion such as that shown in SEQ ID NO: 39, a connecting peptide L1 such as that shown in SEQ ID NO: 23, a shielding portion such as that shown in any one of SEQ ID NO: 29, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 85, a connecting peptide L2 such as that shown in any one of SEQ ID NO: 25, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73, and a cytokine portion such as that shown in SEQ ID NO: 27, SEQ ID NO: 74, or SEQ ID NO: 86. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, an antibody portion that specifically binds to a target, and a sequence selected from any one of SEQ ID NO: 1 or 87.

[0029] In some embodiments, the fusion protein is selected from the antibody fusion proteins shown in Table 1. In a specific embodiment, the fusion protein is the antibody fusion protein ICP-068 or ICP-415 of the present invention.

[0030] In a second aspect, the present invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding the activatable antibody fusion protein according to the first aspect of the present invention. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 34, 36, or 38.

[0031] In a third aspect, the present invention provides a host cell comprising the nucleic acid molecule according to the second aspect of the present invention.

[0032] In some embodiments, the host cell has an altered glycosylation mechanism so that fucose residues are not attached to N-oligosaccharide chains or such attachment is minimized. Preferably, the host cell lacks relevant fucosyltransferase activity or fucose transport activity. In some embodiments, the fucosyltransferase is α1,6-fucosyltransferase (FUT8). In some embodiments, the fucose transporter is GDP-fucose transporter (FUCT1).

[0033] In some embodiments, the host cell is selected from CHO cells, COS cells, HeLa cells, HEK cells, such as HEK 293 cells.

[0034] In a fourth aspect, the present invention provides a method for producing the activatable antibody fusion protein according to the first aspect of the present invention, comprising culturing the host cell according to the third aspect of the present invention to express the fusion protein, and isolating the expressed fusion protein.

[0035] Furthermore, the present invention provides an activatable antibody fusion protein product produced by the method according to the fourth aspect of the present invention, characterized in that the fucosylation level of Asn 297 at position Fc region of the immunoglobulin is reduced, preferably, the activatable antibody fusion protein having fucosylation modification at Asn 297 at position Fc region of the immunoglobulin accounts for 10% or less of the total amount of all activatable antibody fusion proteins, and the amino acid numbering is according to the EU numbering system.

[0036] In some embodiments, in the activatable antibody fusion protein product, the non-fucosylated activatable antibody fusion protein has enhanced antibody-dependent cellular cytotoxicity compared to a fucosylated control fusion protein.

[0037] Furthermore, the present invention provides the use of the activatable antibody fusion protein, nucleic acid molecule, and activatable antibody fusion protein product of the present invention in the preparation of a drug or reagent for diagnosing, treating, or preventing a tumor or autoimmune disease. In some embodiments, the tumor is a tumor associated with CLDN18.2, a tumor associated with HER2, or a tumor associated with CD20. In further embodiments, the tumor is a solid tumor, such as gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, breast cancer; blood cancer, such as lymphoma (such as non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, diffuse large B-cell non-Hodgkin's lymphoma, follicular lymphoma, etc.), leukemia (such as chronic lymphocytic leukemia, etc.). In some embodiments, the autoimmune disease includes, for example, rheumatoid arthritis, autoimmune hemolytic anemia, pure red cell aplasia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura, Evans syndrome, vasculitis (such as granulomatosis with polyangiitis, etc.), bullous skin diseases (such as pemphigus, pemphigoid, etc.).

[0038] The activatable antibody fusion protein of the present invention targets tumor-specific antigens, thereby carrying cytokines to the target tumor location, and activating the activity of cytokines in the tumor microenvironment through tumor-specifically expressed enzymes, achieving effective targeting, while the coupling of Fc and cytokines increases the half-life of cytokines. Further, the present invention uses an activatable antibody fusion protein comprising immunoglobulin Fc, which not only has the antigen targeting effect of the antibody, but also retains the effector function of the Fc portion, including antibody-dependent cell-mediated cytotoxicity (ADCC). Through the synergy of antibody targeting, effector function and cytokine action, a better therapeutic effect is achieved. Therefore, the activatable antibody fusion protein of the present invention represents a promising active pharmaceutical ingredient.

[0039] The following description will use a CLDN18.2 activatable antibody fusion protein as an example. The examples listed are for illustrative purposes only and do not limit the scope of the present invention. Those skilled in the art will appreciate that the concepts of the present invention are also applicable to various tumor-specific antigens and cytokines, and are not limited to their specific sequences.

[0040] In a specific embodiment, the activatable antibody fusion protein of the present invention is mainly composed of an anti-CLDN18.2 antibody and an IL-2 / IL-2Rα complex, and is coupled to IL-2 and IL-2Rα via a cleavable connecting peptide to form an activatable antibody fusion protein; at the same time, the Fc-coupled sugar chain is modified through genetic engineering and cell engineering methods to further enhance the antibody-dependent cellular activity of the fusion protein.

[0041] In one embodiment, an activatable IL-2 is fused to the Fc of a CLDN18.2 antibody via a linker sequence 1 to obtain an activatable antibody fusion protein targeting CLDN18.2, CLDN18.2-Pro-IL2. In general, the activatable antibody fusion protein targeting CLDN18.2 comprises, starting from the N-terminus: the binding region of an antibody targeting CLDN18.2, the Fc fragment of the antibody, a linker sequence 1 linking the Fc fragment and the IL-2 receptor, an interleukin-2 receptor subunit α (IL-2Rα) linked to a first linker, a cleavable linker sequence 2 linked to IL-2, and an interleukin-2 (IL-2) wild-type or IL-2 mutant protein.

[0042] This article describes CLDN18.2-activatable antibody fusion proteins in the form of multifunctional fusion proteins H7E12-2-Pro-IL2 (H7E12-2 antibody, a multifunctional fusion protein of an IL-12 / IL-2Rα conjugate plus an MMP14-cleavable peptide chain), 432-Pro-IL2 (432 antibody, a multifunctional fusion protein of an IL-12 / IL-2Rα conjugate plus an MMP14-cleavable peptide chain), 362-Pro-IL2 (362 antibody, a multifunctional fusion protein of an IL-12 / IL-2Rα conjugate plus an MMP14-cleavable peptide chain), and Hit2.2-Pro-IL2 (Hit2.2 antibody). , IL-12 / IL-2Rα conjugate plus a multifunctional fusion protein of an MMP14-cleavable peptide chain) as an example, using this architectural design in combination with a CLDN18.2 target antibody, the present invention discloses a multifunctional fusion protein that targets Claudin18.2, has optimized Fc function, and has the biological effect of an IL-2 / IL-2Rα complex, obtained by genetic engineering technology. The present invention also discloses the amino acid sequence encoding the multifunctional fusion protein, the architectural design, the recombinant cell containing the recombinant vector, the recombinant cell lacking the fucose modification function containing the recombinant vector, the preparation method of the multifunctional fusion protein, and the medical use of the multifunctional fusion protein.

[0043] definition

[0044] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0045] Provided herein are antibodies (e.g., monoclonal antibodies) that specifically bind to CLDN18.2 and antigen-binding fragments thereof. In a specific aspect, provided herein are monoclonal anti-CLDN18.2 antibodies that specifically bind to CLDN18.2, wherein the anti-CLDN18.2 antibodies include variants of parent antibodies. In a specific aspect, provided herein are antibodies that specifically bind to CLDN18.2 (e.g., human CLDN18.2). The term "CLDN18.2" refers to any CLDN18.2 receptor known to those skilled in the art. For example, the CLDN18.2 can be from a mammal, for example, CLDN18.2 can be from a human or a cynomolgus monkey.

[0046] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0047] Sequence "identity" or "identity" has an art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule (Gribskov & Devereux, 1991; Griffin & Griffin, 1994; Heijne, 1987; Smith, 1994).

[0048] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody, but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains binding specificity and at least partial specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (Welschof & Krauss, 2003). The fragment may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), acquires immunospecific binding (i.e., exhibits at least or at least about 107 -10 8 M -1 "Functional fragments" or "analogs of anti-CLDN18.2 antibodies" are fragments or analogs that prevent or substantially reduce the ability of the receptor to bind to its ligand or initiate signal transduction. As used herein, functional fragments are generally synonymous with "antibody fragments" and, with respect to antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind to its ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, and the like. An "Fv" fragment consists of a dimer (VH-VL dimer) formed by non-covalent association of the variable domains of one heavy chain and one light chain. In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer, as in the case of an intact antibody. These six CDRs collectively confer target binding specificity to the intact antibody. However, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) can still have the ability to recognize and bind to a target.

[0049] As used herein, " monoclonal antibody " refers to the colony of identical antibodies, represents that each independent antibody molecule in the monoclonal antibody colony is identical with other antibody molecules.This characteristic is contrary to the characteristic of the polyclonal colony of antibody, and the polyclonal colony of described antibody comprises the antibody with multiple different sequences.Monoclonal antibody can be prepared by many known methods.For example, monoclonal antibody can be prepared by immortalized B cell, for example, by merging with myeloma cell to produce hybridoma cell line or by using viral infection B cell such as EBV.Recombinant technology can also be used for preparing antibody from the clonal colony of host cell in vitro by transforming host cell with the plasmid of the artificial sequence of nucleotide that carries coding antibody.

[0050] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies with the same domains produced synthetically.

[0051] As used herein, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present on a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present in an amount of about 1×10 7 M -1 or 1x108 M -1 or greater affinity constant Ka (or 1x10 -7 M or 1×10 -8 The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available.

[0052] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0053] As used herein, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0054] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0055] As used herein, a "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the purpose of amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are typically kept episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0056] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0057] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain episomal or that are integrated into the host cell genome.

[0058] As used herein, "activatable antibody fusion protein" refers to a protein in which a complete antibody and a cytokine are fused via a connecting peptide to form a fusion protein, and the cytokine is fused to its receptor via a cleavable linker. On the one hand, the binding of the cytokine receptor to the cytokine reduces the binding activity of the activatable cytokine to normal tissues, and on the other hand, the cleavage of the cleavable linker by metalloproteinases in tumor tissues releases the cytokine from the cytokine receptor.

[0059] Claudin is a key member of the tight junction protein family, playing a crucial role in cell-cell and cell-matrix connections. Claudin18, encoded by the gene CLDN18, is a tetraspanin with two extracellular domains. It is expressed in tissues such as the stomach, pancreas, and lung. Claudin18 is considered a diagnostic marker and therapeutic target (Krause et al., 2008).

[0060] The Claudin18 family includes two variants: Claudin18.1 (CLDN18.1) and Claudin18.2 (CLDN18.2). CLDN18.2 is widely expressed in gastric tumors and has recently been found to be expressed in pancreatic, esophageal, and lung cancers (Jovov et al., 2007; Karanjawala et al., 2008). In normal cells, the tight intercellular junctions prevent antibodies from accessing Claudin18.2. However, in tumor cells, cytopathic changes lead to loose intercellular junctions, exposing CLDN18.2 molecules to which antibodies can bind. Therefore, CLDN18.2 has become an ideal drug target (Klamp et al., 2011).

[0061] Zolbetuximab (IMAB362), a chimeric human IgG1 antibody currently in clinical development, is an existing CLDN18.2 antibody. Results from a Phase IIb clinical study (NCT01630083, FAST 2015) showed that the combination of zolbetuximab with epirubicin, oxaliplatin, and capecitabine (EOX) improved prognosis compared with EOX alone in patients with high CLDN18.2 expression (intensity ≥2+ in ≥70% of tumor cells) (PFS, 9.0 months vs. 5.7 months; HR = 0.38; OS, 16.5 months vs. 8.9 months; HR = 0.50) (Sahin et al., 2021). Currently, at least three monoclonal antibodies, one CLDN18.2-ADC, and one bispecific antibody are in Phase I clinical trials. However, monoclonal antibodies have limited efficacy and high dosages, and ADCs and bispecific antibodies have significant side effects. Therefore, the exploration of novel therapeutic products targeting CLDN18.2 is still needed.

[0062] HER2, or human epidermal growth factor receptor-2 (HER2), is a member of the epidermal growth factor receptor family with tyrosine kinase activity. Dimerization of the HER2 receptor leads to autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor and initiates multiple signal transduction pathways, leading to cell proliferation and carcinogenesis. Amplification or overexpression of HER2 occurs in nearly 15-30% of breast cancer and 10-30% of gastric cancer / gastroesophageal cancer, and serves as a prognostic and predictive biomarker. Overexpression of HER2 has also been seen in other cancers, such as ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer.

[0063] CD20 is a transmembrane protein expressed exclusively on B cells. Although the biological role of this antigen remains to be fully determined, its unique expression profile makes it a promising target for cancer immunotherapy. The anti-CD20 monoclonal antibody rituximab was approved in 1997 for the treatment of non-Hodgkin's lymphoma.

[0064] The cytokine interleukin-2 (IL-2) is essential for the survival and expansion of T cells, especially natural killer CD8+ T cells and NK cells. As used herein, IL-2 "wild" polypeptide refers to a form of IL-2 that is otherwise identical to an IL-2 "mutant", but has wild-type IL-2 amino acids at each amino acid position of the IL-2 mutant. Similarly, as used herein, an IL-2 truncate refers to a form of IL-2 obtained by truncating one or more amino acids from the C- and / or N-terminus of a wild-type or mutant IL-2, and as used herein, an IL-2 mutant refers to an IL-2 "wild" polypeptide that is otherwise identical to an IL-2 "mutant" polypeptide, having an amino acid different from the wild-type at each amino acid position.

[0065] IL-2 is mainly secreted by activated CD4+ helper T cells. By binding to the IL-2Rβγ dimer receptor on CD8+ T cells or NK cells, it promotes the downstream JAK1 / JAK3-STAT5 signaling pathway and promotes the survival of T cells and NK cells. In addition, IL-2 is also necessary for the maintenance of regulatory T cells (Treg). Treg expresses high-affinity IL-2Rαβγ trimer receptors with an affinity of IL-2 of Kd ~ 10 -11 M, affinity of IL-2Rβγ dimer receptor to IL-2, Kd ~ 10 -9 M, affinity of IL-2Rα monomer to IL-2, Kd ~ 10 -8 M (Hernandez et al., 2022).

[0066] IL-2Rα, also known as CD25, is one of the receptors for IL-2. It participates in the regulation of immune tolerance by controlling the activity of regulatory T cells (Tregs), which in turn inhibit the activation and expansion of autoreactive T cells. IL-2Rα and IL-2 have a relatively low affinity, with a dissociation constant, Kd, ​​of approximately 10 nM. The extracellular domain of IL-2Ra resembles a bent arm, with D1 and D2, located at the N-terminus and C-terminus, forming a 90° angle. D1 and D2 are connected by 42 amino acid residues, while the C-terminus of D2 is connected to the transmembrane domain by 54 amino acid residues. IL-2Rα truncations are otherwise identical to the "wild-type" IL-2 polypeptide, except for truncations of varying lengths of amino acids at the C-terminus.

[0067] As used herein, "immune cells" refer to cells involved in or associated with immune responses, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. For example, the immune cells include T lymphocytes, B lymphocytes, NK cells, etc.

[0068] "Defucosylation" as used herein refers to the natural lack of fucose transporters or fucosyltransferases in host cells, or the knockout or reduction of fucose transporters or fucosyltransferases by gene editing methods, so that the human IgG antibody expressed by the host cell, or the N-glycan at the Asn-297 site of the IgG Fc segment containing it, loses or significantly reduces the ability of core fucosylation, thereby enhancing its affinity for Fcγ receptors and ADCC activity.

[0069] As used herein, "pharmaceutical composition" refers to a pharmaceutically acceptable composition comprising, for example, one or more, e.g., two, three, four, five, six, seven, eight, or more, of the therapeutic agents described herein, formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0070] As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or progression of the disease. Treatment also includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided, as well as the compositions provided herein.

[0071] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG1 shows the structure of an activatable antibody fusion protein, wherein:

[0073] A) Schematic diagram showing the structure and function of activatable antibody fusion proteins;

[0074] B) Schematic diagram showing the structure of the activatable-cytokine fusion protein.

[0075] FIG2 shows an SDS-PAGE image of the CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:

[0076] A) shows the SDS-PAGE image of the activatable IL2 fusion protein and CLDN18.2 antibody;

[0077] B) shows the SDS-PAGE image of CLDN8.2-Pro-IL2 activatable antibody fusion protein;

[0078] C) SDS-PAGE showing ADCC-enhanced CLDN8.2-Pro-IL2;

[0079] D) shows the SDS-PAGE image of CLDN8.2-Pro-IL2 digested with protease MMP14;

[0080] E) shows the SDS-PAGE images of antibody fusion proteins with different linker peptides;

[0081] F) SDS-PAGE image of antibody fusion protein with enhanced ADCC of novel linker peptide.

[0082] FIG3 shows the SEC-HPLC chart of the CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:

[0083] A) shows the SEC-HPLC graph of activatable IL2 fusion protein and CLDN18.2 antibody;

[0084] B) shows the SEC-HPLC profile of activatable IL2 and antibodies;

[0085] C) shows the SEC-HPLC graph of the activatable IL2 antibody fusion protein with enhanced ADCC;

[0086] D) shows the SEC-HPLC profile of antibody fusion proteins with different linker peptides;

[0087] E) shows the SEC-HPLC profile of the antibody fusion protein with enhanced ADCC by the novel linker peptide.

[0088] FIG4 shows the glycoforms of the CLDN18.2-Pro-IL-2 antibody fusion protein in fucose-knockout host cells, wherein:

[0089] A) Glycoform analysis of the fusion protein ICP-130 expressed by H7E12-Pro-IL2 in α1,6-fucosyltransferase (FUT8) knockout CHO-K1 host cells;

[0090] B) shows the effect of H7E12-Pro-IL2 on SLC35C1 knockout (FUCT1 deletion) CHO-K1-GFT - Glycoform analysis of the fusion protein ICP-155 expressed in (CHOK1-AF) host cells.

[0091] FIG5 shows the detection of the binding ability of CLDN18.2-Pro-IL-2 antibody fusion protein to IL-2 receptor.

[0092] FIG6 shows a reducing SDS-PAGE image of the in vitro MMP14 cleavage of the CLDN18.2-Pro-IL-2 antibody fusion protein.

[0093] FIG7 shows the effect of antibody fusion protein mutants on NK-92 cell proliferation, wherein:

[0094] A) shows the effects of active and inactive forms of ICP-302 and ICP-303 on NK-92 cell proliferation;

[0095] B) shows the effects of ICP-414, ICP-415, ICP-416, and ICP-417 in active and inactive forms on NK-92 cell proliferation;

[0096] C) shows the effects of active and inactive forms of ICP-198 on NK-92 cell proliferation.

[0097] FIG8 shows the effect of antibody fusion protein mutants on CTLL-2 cell proliferation, wherein:

[0098] A) shows the effects of active and inactive forms of ICP-302 and ICP-303 on the proliferation of CTLL-2 cells;

[0099] B) shows the effects of active and inactive forms of ICP-414, ICP-415, ICP-416, and ICP-417 on the proliferation of CTLL-2 cells;

[0100] C) shows the effects of active and inactive forms of ICP-198 on the proliferation of CTLL-2 cells.

[0101] FIG9 shows the effects of active and inactive forms of the CLDN18.2-Pro-IL-2 antibody fusion protein on IFN-γ release by PBMC-derived T cells, wherein:

[0102] A) shows the effects of active and inactive forms of ICP-070 on IFN-γ release by PBMC-derived T cells;

[0103] B) shows the effects of active and inactive forms of ICP-087 on IFN-γ release by PBMC-derived T cells;

[0104] C) shows the effects of active and inactive forms of ICP-068 on IFN-γ release by PBMC-derived T cells;

[0105] D) shows the effects of active and inactive forms of ICP-106 on IFN-γ release from PBMC-derived T cells.

[0106] FIG10 shows the ADCC activity detection of primary NK cells mediated by active and inactive forms of defucosylating CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:

[0107] A) shows the comparison of ADCC activity between ICP-087 and its monoclonal antibody ICP-038;

[0108] B) shows the comparison of ADCC activity between ICP-106 and its mAb ICP-063.

[0109] FIG11 shows the ADCC reporter cell line activity detection mediated by the ADCC-enhanced CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:

[0110] A) shows the comparison of ADCC activities of ICP-070 (wild-type Fc) and its corresponding ADCC-enhanced fusion proteins ICP-087 (defucose), ICP-155 (defucose) and ICP-154 (S239D, I332E);

[0111] B) shows the comparison of ADCC activity between ICP-068 (wild-type Fc) and its corresponding ADCC-enhanced ICP-106 (defucose) and ICP-153 (S239D, I332E) fusion proteins;

[0112] C) shows the ADCC activity of ICP-415 (wild-type Fc) and its corresponding ADCC-enhancing mutations ICP-501, ICP-502, ICP-503, and ICP-198 (defucosylated) antibody fusion proteins in CD16 / V158 cells;

[0113] D) shows the ADCC activity of ICP-415 (wild-type Fc) and its corresponding ADCC-enhancing mutations ICP-501, ICP-502, ICP-503, and ICP-198 (defucosylated) antibody fusion proteins in CD16 / F158 cells;

[0114] FIG12 shows the detection of the effects of active and inactive forms of CLDN18.2-Pro-IL-2 antibody fusion protein on STAT5 phosphorylation signals in NK-92 cells, wherein:

[0115] A) shows the effects of active and inactive forms of ICP-070 on STAT5 phosphorylation signals in NK-92 cells;

[0116] B) shows the effects of active and inactive forms of ICP-087 on STAT5 phosphorylation signals in NK-92 cells;

[0117] C) shows the effects of active and inactive forms of ICP-068 on STAT5 phosphorylation signals in NK-92 cells;

[0118] D) shows the effects of active and inactive forms of ICP-106 on STAT5 phosphorylation signals in NK-92 cells.

[0119] FIG13 shows the tumor suppressive activity assay of ICP-024 and the control drug ICP-015 in the mouse colon cancer model MC-38-hCLDN18.2-A11 cells.

[0120] FIG14 shows the individual tumor growth curves of mice in each drug administration group in the drug efficacy experiment described in FIG13 , wherein:

[0121] A) shows the growth curve of individual tumors in mice of the control group;

[0122] B) shows the individual tumor growth curve of mice in the ICP-024 administration group;

[0123] C) shows the individual tumor growth curve of mice in the ICP-025 administration group;

[0124] D) shows the tumor growth curve of individual mice in the ICP-015-administered group.

[0125] FIG15 shows the tumor suppressive activity test of ICP-414 and other drug-treated groups in MC-38-hCLDN18.2 cells, a mouse colon cancer model, wherein:

[0126] A) shows the growth curve of individual tumors in each group of mice;

[0127] B) shows the growth curve of individual tumors in mice in the control and drug-treated groups;

[0128] C) shows the growth curve of individual tumors in mice in the ICP-414 administration group.

[0129] FIG16 shows the tumor suppressor activity test of ICP-415 and other drug-treated groups in MC-38-hCLDN18.2 cells, a mouse colon cancer model, wherein:

[0130] A) shows the growth curve of individual tumors in each group of mice;

[0131] B) shows the growth curve of individual tumors in mice in the control and drug-treated groups;

[0132] C) shows the tumor growth curve of individual mice in the ICP-4415 administration group.

[0133] FIG17 shows the tumor suppressive activity test of ICP-416 and other drug-treated groups in MC-38-hCLDN18.2 cells, a mouse colon cancer model, wherein:

[0134] A) shows the growth curve of individual tumors in each group of mice;

[0135] B) shows the growth curve of individual tumors in mice in the control and drug-treated groups;

[0136] C) shows the growth curve of individual tumors in mice in the ICP-416-administered group.

[0137] FIG18 shows the tumor suppressive activity assay of ICP-024 and control drug ICP-015 in mouse colon cancer model CT-26-hCLDN18.2 cells.

[0138] FIG19 shows the individual tumor growth curves of mice in each drug administration group in the drug efficacy experiment described in FIG18 , wherein:

[0139] A) shows the growth curve of individual tumors in mice of the control group;

[0140] B) shows the individual tumor growth curve of mice in the ICP-024 administration group;

[0141] C) shows the individual tumor growth curve of mice in the ICP-025 administration group;

[0142] D) shows the tumor growth curve of individual mice in the ICP-015-administered group.

[0143] FIG20 shows the flow cytometry determination of the absolute count of peripheral blood lymphocytes in mice at the end point of the efficacy experiment described in FIG18 , wherein:

[0144] A) shows the absolute numerical changes of peripheral blood CD45+ cells in each drug-treated group;

[0145] B) shows the absolute numerical changes of peripheral blood CD3+ cells in each drug-treated group;

[0146] C) shows the absolute numerical changes of peripheral blood CD8+ cells in each drug-treated group;

[0147] D) shows the absolute numerical changes in peripheral blood CD4+ cells in each drug-treated group.

[0148] FIG21 shows the tumor suppressive activity test of different CLDN18.2 antibody fusion proteins and the control drug ICP-069 in the mouse colon cancer model MC-38-hCLDN18.2-A11 cells.

[0149] FIG22 shows the individual tumor growth curves of mice in each drug administration group in the drug efficacy experiment described in FIG21 above, wherein:

[0150] A) shows the growth curve of individual tumors in mice of the control group;

[0151] B) shows the individual tumor growth curve of mice in the ICP-070 administration group;

[0152] C) shows the individual tumor growth curve of mice in the ICP-068 administration group;

[0153] D) shows the individual tumor growth curve of mice in the ICP-069 administration group;

[0154] E) shows the individual tumor growth curve of mice in the ICP-024 administration group;

[0155] F) shows the tumor growth curve of individual mice in the ICP-087 administration group.

[0156] FIG23 depicts the tumor restimulation and immune memory formation experiments in mice with ICP-024 and ICP-087 tumor regression in the efficacy experiments described in FIG22 .

[0157] FIG24 shows the individual tumor growth curves of mice in each dosing group in the tumor restimulation experiment of ICP-024 and ICP-087 tumor regression mice described in FIG23 , wherein:

[0158] A) shows the growth curve of individual tumors in mice of the control group;

[0159] B) shows the individual tumor growth curve of mice in the ICP-024 administration group;

[0160] C) shows the tumor growth curve of individual mice in the ICP-087 administration group.

[0161] FIG25 shows that compared with Fc-IL-2, the CLDN18.2-Pro-IL-2 antibody fusion protein did not cause capillary leakage and had good peripheral safety.

[0162] Figure 26 shows SDS-PAGE images of different antibody fusion proteins, wherein:

[0163] A) shows the SDS-PAGE image of ICP-269 antibody fusion protein;

[0164] B) shows the SDS-PAGE image of ICP-270 antibody fusion protein.

[0165] Figure 27 shows SEC-HPLC graphs of different antibody fusion proteins, wherein:

[0166] A) shows the SEC-HPLC profile of ICP-269 antibody fusion protein;

[0167] B) shows the SEC-HPLC chart of ICP-270 antibody fusion protein.

[0168] FIG28 shows an SDS-PAGE image of different antibody fusion proteins cleaved by MMP14 in vitro, wherein:

[0169] A) Non-reduced images of MMP14 enzyme cleavage of ICP-269 and ICP-270 antibody fusion proteins;

[0170] B) ICP-269 and ICP-270 antibody fusion protein MMP14 enzyme reduction diagram

[0171] FIG29 shows the binding ability of different antibody fusion proteins to their target antigens, wherein:

[0172] A) shows the binding of ICP-269 to Her2-high-expressing cells BT474;

[0173] B) shows the binding of ICP-270 to CD20-high-expressing REC-1 cells.

[0174] FIG30 shows the effects of different antibody fusion proteins on T cell proliferation, wherein:

[0175] A) shows the effects of ICP-269 and ICP-270 on NK-92 cell proliferation;

[0176] B) shows the effects of ICP-269 and ICP-270 on the proliferation of CTLL-2 cells. Specific implementation plan

[0177] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0178] The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents, raw materials, etc. used in the following examples are commercially available products unless otherwise specified. Zolbetuximab (IMAB362): Ganymed Pharmaceuticals, see NW_004504382.1. Trastuzumab (Trastuzumab), trade name ( Trastuzumab (HER2) is a recombinant DNA-derived humanized monoclonal antibody developed by Roche in Switzerland. It contains a human IgG1 subtype framework and a complementarity-determining region derived from a mouse anti-p185HER2 antibody. It specifically targets the extracellular subdomain IV of the human epidermal growth factor receptor-2 (HER2), competitively blocking the binding of human epidermal growth factor to HER2, thereby inhibiting tumor cell growth. The product was first approved for marketing by the US FDA on September 25, 1998, and was imported into China in 2002. The US patent for trastuzumab expired in June 2019. The applicants of the present invention, with reference to the anti-HER2 monoclonal antibody data and sequences disclosed in Carter, P. and L. Presta, et al. (1992). "Humanization of an anti p185HER2 antibody for human cancer therapy." Proc. Natl. Acad. Sci. USA 89(10): 4285-9., constructed the light and heavy chain variable region and constant region genes of the anti-HER2 humanized antibody and independently constructed the Trastuzumab antibody. Rituximab, trade name RITUXAN, is a human-mouse chimeric monoclonal antibody synthesized using genetic engineering technology. It consists of a human IgG1 kappa constant region and a murine CD20 antibody variable region. It can specifically bind to the transmembrane protein CD20 on the surface of B cells and kill CD20-positive B lymphocytes through two pathways: antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Rituximab was originally developed by Roche Pharma (Schweiz) Ltd., received US FDA approval in 1997, and was launched in China in 2000. The Chinese patent for rituximab injection expired in 2013. The applicants of the present invention independently constructed the rituximab antibody with reference to US Pat. No. 5,736,137.

[0179] Example 1: Preparation of CLDN18.2-Pro-IL-2 Antibody Fusion Protein

[0180] The activatable antibody fusion protein increases the targeting of the cytokine interleukin-2 (IL-2) by binding to the antibody, and prolongs the half-life of the cytokine by fusion with the antibody Fc. At the same time, the interleukin-2 receptor subunit α (IL-2Rα) is fused with IL2 through a cleavable linker and inhibits the activity of IL-2, which reduces the binding activity of interleukin-2 (IL-2) to normal tissues. When it reaches the tumor, the metalloproteinase in the tumor tissue cuts the cleavable linker and releases IL-2 from the IL-2 receptor to subjects in need.

[0181] In another embodiment, the present invention includes a method for reducing the binding activity of activatable interleukin-2 (IL-2) to normal tissues and targeting cancer cells, comprising administering an effective amount of an activatable interleukin-2 (IL-2) fusion protein comprising: interleukin-2 (IL-2) wild type or mutant protein or truncation; a cleavable linker connected to IL-2; an interleukin-2 receptor binding region (IL-2α or IL-2β receptor) or mutant protein or truncation connected to a cleavable linker; and a half-life extender connected to IL-2 or IL-2 receptor, wherein cleavage of the cleavable linker releases IL-2 from the IL-2 receptor to a subject in need thereof.

[0182] Here, activatable IL2 is fused to the Fc of the CLDN18.2 antibody via linker 1 to obtain an activatable antibody fusion protein targeting CLDN18.2, CLDN18.2-Pro-IL2. In general, the activatable antibody fusion protein targeting CLDN18.2 comprises, starting from the N-terminus: the binding region of the antibody targeting CLDN18.2, the Fc fragment of the antibody, linker 1 connecting the Fc fragment and the IL-2 receptor, interleukin-2 receptor subunit α (IL-2Rα) or mutant or truncated IL-2Rα connected to linker 1, a cleavable linker 2 connected to IL-2, and interleukin-2 (IL-2) wild type or IL-2 mutant protein or IL-2 truncation.

[0183] Preferably, the heavy chain HC of the humanized monoclonal antibody H7E12-2 against CLDN18.2 is fused sequentially with linker 1, interleukin-2 receptor subunit α, cleavable sequence 2, and interleukin-2 (IL-2), corresponding to the amino acid sequence SEQ ID NO: 17, and co-expressed with the light chain amino acid sequence SEQ ID NO: 15 to obtain the antibody fusion protein H7E12-2-Pro-IL2, protein number ICP-070.

[0184] Preferably, the heavy chain HC of Zolbetuximab (362) (gene NW_004504382.1) is fused with linker 1, interleukin-2 receptor subunit α, cleavable sequence 2, and interleukin-2 (IL-2) in sequence, and the corresponding amino acid sequence SEQ ID NO: 13 and the light chain amino acid sequence SEQ ID NO: 11 are co-expressed to obtain the antibody fusion protein 362-Pro-IL2, protein number ICP-069.

[0185] Preferably, the heavy chain HC of the human CLDN18.2 antibody 432 is fused with linker 1, interleukin-2 receptor subunit α, cleavable sequence 2, and interleukin-2 (IL-2) in sequence, and the corresponding amino acid sequence SEQ ID NO: 9 and the light chain amino acid sequence SEQ ID NO: 7 are co-expressed to obtain the antibody fusion protein 432-Pro-IL2, protein number ICP-068.

[0186] Preferably, the heavy chain HC of the human CLDN18.2 antibody Hit2.2 is fused with linker 1, interleukin-2 receptor subunit α, cleavable sequence 2, and interleukin-2 (IL-2) in sequence, and the corresponding amino acid sequence SEQ ID NO: 5 and the light chain amino acid sequence SEQ ID NO: 3 are co-expressed to obtain the antibody fusion protein Hit2.2-Pro-IL2, protein number ICP-024.

[0187] More preferably, the human CLDN18.2 antibody 432 heavy chain HC is sequentially fused with linker 1, interleukin-2 receptor subunit α mutant 1, mutant 2, or mutant 3, linker 3, linker 4, linker 5, and interleukin-2 (IL-2), wherein the amino acid sequence of interleukin-2 receptor subunit α mutant 1, mutant 2, or mutant 3 is as shown in SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83; the amino acid sequence of linker 3, linker 4, linker 5 is as shown in SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73. Activatable antibody fusion protein molecules ICP-302, ICP-303, ICP-414, ICP-415, ICP-416, and ICP-417 are thus obtained, with corresponding heavy chain amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59.

[0188] Interleukin-2 receptor subunit α (IL-2Rα) (SEQ ID NO: 27) was linked to the Fc region of an antibody via a first linker sequence (SEQ ID NO: 23), and interleukin-2 (IL-2) was then linked to the interleukin-2 receptor binding region via a second cleavable linker (SEQ ID NO: 25) to create the fusion protein ICP-015 (SEQ ID NO: 1). These linkages were achieved by DNA homologous recombination.

[0189] After IL-2Rα and IL-2 are linked, IL2 binds to IL-2Rα, reducing its toxicity in the heart, lungs, kidneys, or central nervous system. On the other hand, the cleavable linker linked to IL2 is cleaved by the protease MMP14, which is upregulated in the tumor microenvironment, and released from its interleukin-2 receptor binding region, thereby concentrating at the tumor site. At the same time, the antibody Fc region can prolong the half-life of IL-2. In general, the fusion protein contains: (1) target protein binding sequence; (2) human IgG1 Fc segment; 3) linker 1; (4) human IL-2Rα or mutant protein; (5) cleavable linker 2, linker 3 or linker 4 or linker 5; (6) human IL-2; or (1) human IgG1 Fc segment; (2) linker 1; (3) human IL-2Rα; (4) cleavable linker 2; (5) human IL-2. By linking with different target protein binding regions, the IL2 fusion protein ICP-015 shown in the table below and various activatable antibody fusion proteins are obtained. The sequences and expression host cells are shown in Table 1.

[0190] Table 1: Summary of antibody and fusion protein sequences and host cells

[0191] Example 2: Characterization of CLDN18.2-Pro-IL-2 Antibody Fusion Protein

[0192] CHO-S cells were cultured at 37°C, 8% CO2, and 100 rpm to a cell density of 6 × 10 6 The constructed vectors were transfected into the above cells using lipofectamine at a transfection plasmid concentration of 1 mg / ml. The lipofectamine concentration was determined according to the ExpiCHO™ Expression System kit. The cells were cultured at 32°C, 5% CO2, and 100 rpm for 7-10 days. Feeds were performed 18-22 hours after transfection and again on day 5. The culture products were centrifuged, filtered through a 0.22 μm filter, and the supernatant was collected. The antibody or fusion protein was purified using Protein A and an ion column.

[0193] The specific operating steps for ProteinA and ion column purification are as follows: after high-speed centrifugation of the cell culture fluid, the supernatant is taken and affinity chromatography is performed using Cytiva's ProteinA chromatography column. The equilibration buffer used for chromatography is 1×PBS (pH7.4). After the cell supernatant is loaded and bound, it is washed with PBS until the ultraviolet light returns to the baseline, and then the target protein is eluted with 0.1M glycine (pH3.0) as an elution buffer, and the pH is adjusted to neutral for storage using Tris. The pH of the product obtained by affinity chromatography is adjusted to 1-2 pH units lower or higher than pI, and appropriately diluted to control the sample conductivity below 5ms / cm. Using appropriate corresponding pH buffers such as phosphate buffer, acetate buffer and other conditions, conventional ion exchange chromatography methods in the art, such as anion exchange or cation exchange, perform NaCl gradient elution under corresponding pH conditions, and select the collection tube where the target protein is located based on UV280 absorption and store it together.

[0194] The purified eluate was then ultrafiltered and exchanged into a buffer solution. Protein purity and content were determined by SDS-PAGE gel electrophoresis, as shown in Figure 2.

[0195] The purity of the fusion protein was further determined by SEC-HPLC. The results showed that the target antibody or fusion protein had a purity of more than 90% after one-step purification, which was relatively high, as shown in Figure 3A-E and Table 2.

[0196] Table 2: Purity results of each antibody fusion protein

[0197] Example 3: Preparation and glycoform identification of ADCC-enhanced CLDN18.2-Pro-IL-2 activatable antibody fusion protein

[0198] The expression of H7E12-2-Pro-IL2 in FUT8 knockout CHO-K1 / FUT8- cells was performed by Nanjing Pengbo Biotechnology Co., Ltd. The cell line was passaged in Expi-CHOS expression medium for production at a cell density of 0.2-0.3 x 10 6 / mL, subculture cycle 2 to 3 days. Dilute cells to 2x 10 6 / mL. On the day of transfection, the cell density should be 6 x 10 6 / mL, with a viability greater than 95%. On the day of transfection, transfection was performed according to the transfection kit's instructions. 7E12-2-pro-IL2 heavy and light chains were used at 100 μg, with a light chain to heavy chain molar ratio of 1:1. On the first day after transfection, Enhancer and Expichos Feed medium were added, and the cells were cooled to 32°C and incubated. On the fifth day after transfection, Expichos Feed medium was added. Cell viability was monitored 10 to 14 days after transfection. When viability was less than 70%, cells were harvested and protein was purified using the ICP-130 designation. Protein expression and purity are shown in Figures 2 and 3 and Table 2.

[0199] The expressed fusion protein ICP-130 was quantitatively analyzed for glycoforms using fluorescence-labeled chromatography and mass spectrometry, and the results are shown in Figure 4 and Table 3. The results showed that the major glycoforms G0F and G1F modified with fucose in the Fc region were below the detection limit.

[0200] H7E12-2-Pro-IL2 and 432-Pro-IL2 in SLC35C1 knockout (FUCT1 deficiency) CHO-K1 / GFT cells - Expression in CHOK1-AF cells was performed by Beijing Huafang Tianshi Biopharmaceutical Co., Ltd. The cell line was cultured in Transpro CD01 for passage. Transfection was performed using PEI in a total volume of 200 mL. The vectors used were H7E12-2-pro-IL2 or 432-Pro-IL2 heavy and light chains, with a dosage of 200 μg and a concentration of 1.6 μg / ml. The molar ratio of light chain to heavy chain was 1:1. The cell density was adjusted to 5x10 before transfection. 6 / mL, then prepare the transfection complex, mix the calculated plasmid and PEI, react for 10 minutes, and add to the prepared cells. Culture at 37°C, 5% CO2, and shake at 125 rpm. On the first day after transfection, add sodium butyrate and 0.1g / L dextran sulfate sodium salt, and cool to 32°C for culture. On days 1, 3, 5, 7, and 9 after transfection, perform fed-batch culture with 3% MaxFeed TM 403A1 (Dongning Bio) and 0.3% MaxFeed TM 403B1 (Dongning Biotechnology) was fed-through. From day 10 to 14 after transfection, cell viability and glucose content were monitored, and glucose was added if insufficient. Cells were harvested after 14 days of culture, and protein was purified. Protein expression and purity are shown in Figures 2 and 3 and Table 2.

[0201] The expressed fusion protein ICP-155 was quantitatively analyzed for glycoforms using fluorescence-labeled chromatography and mass spectrometry, and the results are shown in Figure 4B and Table 4. The results showed that the major glycoforms G0F and G1F modified with fucose in the Fc region were below the detection limit.

[0202] Table 3: Glycoform analysis of antibody fusion protein ICP-130

[0203] Table 4: Glycoform analysis of antibody fusion protein ICP-155

[0204] Example 4: Determination of the Binding Ability of CLDN18.2-Pro-IL-2 Antibody Fusion Protein to IL-2Rα and IL-2Rβγ

[0205] HEK-Blue TM IL-2 cells (purchased from Invivogen) overexpressed human CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ) genes, thus expressing low-affinity IL-2Rα receptors, medium-affinity IL-2Rβγ receptors, and high-affinity IL-2Rαβγ receptors on the cell membrane. 8 x 10 4 Cells were incubated with different CLDN18.2-Pro-IL-2 fusion proteins at room temperature for 30 minutes, starting at a 20 nM concentration and followed by 5-fold dilutions across nine concentration gradients. Following incubation, the cells were washed once with flow cytometry buffer, and a PE-labeled anti-human IgG Fc secondary antibody was added. The cells were incubated at room temperature for 30 minutes, washed once with flow cytometry buffer, and the mean fluorescence intensity was measured using a NovoCyte Quanteon flow cytometer (Agilent). Data were analyzed using GraphPad Prism 7.0 software, and dose-effect curves were fitted using nonlinear S-curve regression to generate the data, from which the EC values ​​were calculated. 50 The results are shown in Figure 5 and Table 5.

[0206] ICP-159 is a fusion protein of human IgG1 Fc and IL-2 expressed in series. It serves as a positive control for IL-2 receptor binding. TM The binding strength EC50 on IL-2 cells was 0.74 nM, while the different CLDN18.2-Pro-IL-2 fusion proteins did not bind to HEK-Blue due to IL-2-fold IL-2R α shielding. TM The IL-2 receptors expressed on IL-2 cells were bound, proving that the shielding effect was good and achieved the effect of inhibiting the binding of IL-2 to the receptors.

[0207] Table 5: Detection of the binding ability of CLDN18.2-Pro-IL-2 antibody fusion protein to IL-2 receptor

[0208] N / A: Not detected.

[0209] Example 5: In vitro MMP14 cleavage of CLDN18.2-Pro-IL-2 antibody fusion protein

[0210] hrFurin-activated proenzyme MMP-14 was diluted in 1X activation buffer (50 mM Tris-HCl pH 9.0, 1 mM CaCl2, 0.5% Brij-35) and pre-activated at 37°C for 2 hours to activate the MMP14 enzyme. Fusion proteins ICP-068, ICP-070, ICP-087, and ICP-106 to be cleaved were cleaved at an equimolar concentration of 1.2 μmol / L in 10X cleavage buffer (500 mM Tris-HCl pH 7.5, 30 mM CaCl2, 10 μM ZnCl2) and incubated at 37°C for 48 hours. A control group was also established, without MMP14 enzyme, using 1X activation buffer instead of MMP14 enzyme. Enzymatic cleavage efficiency was verified by SDS-PAGE. A 10 μL sample from each digestion system was mixed with 3.3 μL of 4X loading buffer containing DTT and incubated at 95°C for 10 minutes. A 2 μg sample was loaded onto a 15-well precast gel and electrophoresed at a constant voltage of 100 V for 100 minutes. Staining and destaining were performed using an eStain L1 protein stainer (GenScript). Enzyme digestion results are shown in Figure 6. MMP14 cleavage of the fusion protein releases active IL-2 protein, approximately 15 kDa. The biological activity of the fusion protein was subsequently assessed after MMP14 digestion (+) or non-cleavage (-).

[0211] Example 6: Detection of NK-92 and CTLL-2 Cell Proliferation Activity of CLDN18.2-Pro-IL-2 Antibody Fusion Protein

[0212] Both the human NK cell line NK-92 (CRL-2407, ATCC) and the mouse T lymphocyte line CTLL-2 (purchased from the Institute of Biophysics, Chinese Academy of Sciences) are IL-2-dependent. The proliferative activity of MMP14-cleaved (+) or uncleaved (-) fusion proteins was evaluated in vitro using an IL-2-starved NK-92 and CTLL-2 cell model. 3000 cells / well were plated in a 96-well plate. The active and inactive fusion proteins were diluted fivefold at a starting concentration of 26 nM over nine concentrations. A PBS negative control was also included. After incubation, the flat-bottom 96-well plate was removed and equilibrated to room temperature. 30 μL of CTG-Glo reagent (Promega, Madison, WI) was added and thoroughly lysed with shaking for 10 minutes. After standing at room temperature for 10 minutes, 50 μL of the supernatant was transferred to a flat-bottom 384-well plate and the fluorescence signal was measured using an Envision Multi-Mode Microplate Reader (Perkin Elmer, Waltham, MA). Data were analyzed using GraphPad Prism 7.0 software, and nonlinear S-curve regression was used to fit the data to obtain the dose-effect curve, from which the EC was calculated. 50 EC values ​​of NK-92 (n=3) and CTLL2 (n=3) cells 50 ± SEM are summarized in Table 6. Fold changes ± SEM are from uncut (-) EC 50 / Cutting form (+)EC 50 Calculated.

[0213] Table 6: Summary of cell proliferation activity of CLDN18.2-Pro-IL-2 antibody fusion protein in NK-92 and CTLL-2 cells

[0214] N / A, not detected, data are shown as mean ± SEM.

[0215] The fusion proteins ICP-068, ICP-106, ICP-070 and ICP-087 were cleaved by MMP and expressed as ICP-068+, ICP-106+, ICP-07+ and ICP-087+, respectively; the uncleaved ICP-068, ICP-106, ICP-070 and ICP-087 were expressed as ICP-068-, ICP-106-, ICP-070- and ICP-087-, respectively. The results showed that the CLDN18.2 monoclonal antibodies H7E12-2 and 432 had no in vitro activity in promoting the proliferation of NK-92 and CTLL-2 cells. The fusion proteins ICP-068+, ICP-106+, ICP-07+ and ICP-087+ had no in vitro activity in promoting the proliferation of NK-92 and CTLL-2 cells. 50The EC values ​​of human recombinant IL-2 were 17.7±5.2nM, 20.7±8.2nM, 7.5±1.7nM, and 12.9±3.1nM, respectively. 50 Activity 13.5 ± 1.5 equivalent; EC of uncleaved ICP-068-, ICP-106-, ICP-070-, and ICP-087- in NK-92 cells 50 The EC values ​​of the inactive and active forms of the fusion proteins ICP-068, ICP-106, ICP-070, and ICP-087 were 419.0 ± 78.9 nM, 157.0 ± 39.7 nM, 296.3 ± 57.0 nM, and 154.7 ± 26.8 nM, respectively. 50 The fold changes were 26.6±5.0 times, 8.5±1.3 times, 40.0±2.6 times, and 12.6±2.2 times, respectively. The results showed that the activity of cytokine IL-2 in the shielded CLDN18.2-Pro-IL-2 fusion protein was significantly reduced. After MMP14 cleavage, IL-2 could be effectively released to promote the expansion of NK cells.

[0216] In CTLL-2 cells, ECs of fusion proteins ICP-068+, ICP-106+, ICP-070+, and ICP-087+ 50 The EC values ​​of human recombinant IL-2 were 12.0±2.1nM, 8.1±1.9nM, 6.1±2.0nM, and 6.2±1.9nM, respectively. 50 Activity 10.3 ± 4.7 equivalent; uncleaved ICP-068-, ICP-106-, ICP-070-, and ICP-087-EC in CTLL-2 cells 50 EC values ​​of the inactive and active forms of the fusion proteins ICP-068, ICP-106, ICP-070, and ICP-087 in CTLL-2 cells were 201.3 ± 66.3 nM, 59.0 ± 17.0 nM, 77.7 ± 1.5 nM, and 68.5 ± 26.5 nM, respectively. 50 The fold changes were 17.4±4.8 times, 7.2±0.4 times, 15.7±4.6 times, and 9.0±0.6 times, respectively. Similar to the results of NK-92 cells, the activity of cytokine IL-2 in the shielded form of CLDN18.2-Pro-IL-2 fusion protein was significantly reduced in CTLL-2 mouse T cells. After MMP14 cleavage, IL-2 could be effectively released to promote the expansion of T cells.

[0217] The NK-92 and CTLL-2 cell proliferation activity was also tested using antibody fusion proteins composed of different IL-2Ra mutants or IL-2 mutants, such as ICP-302, ICP-303, ICP-414, ICP-415, ICP-416, and ICP-417, as shown in Figures 7, 8, and Table 7. The results showed that the inactive forms and active forms of the fusion proteins ICP-414, ICP-415, ICP-ICP-302, and ICP-303 played a significant role in the proliferation of NK-92 cells. 50 The fold changes were 38-fold, 97-fold, 40.5±19.61-fold, and 11.13±4.91-fold, respectively. The results showed that the CLDN18.2-Pro-IL-2 fusion protein mutant also significantly reduced cytokine IL-2 activity. After MMP14 cleavage, it could effectively release IL-2, promote the expansion of NK cells, and have comparable or superior prodrug effects. In CTLL-2 cells, the fold changes were 3.3, 3.4, 5.83±2.71, and 2.03±0.32, respectively, also demonstrating a certain prodrug effect.

[0218] Table 7: Summary of EC50 and fold change of antibody fusion protein mutants on NK-92 cell and CTLL-2 cell proliferation

[0219] Activity assays on both cell lines demonstrated that the uncleaved prodrug form blocks IL-2 activity. When the masking peptide is cleaved, active IL-2 is released, effectively promoting T and NK cell proliferation. The difference in activity between the prodrug and active drug on NK-92 cells ranged from 11 to 97 times.

[0220] Example 7: Detection of IFN-γ Release from Primary T Cells Using CLDN18.2-Pro-IL-2 Antibody Fusion Protein

[0221] IL-2 is essential for maintaining the survival of primary T cells, so the effect of CLDN18.2-Pro-IL-2 fusion protein on the homeostasis and IFN-γ release of primary T cells was tested. PBMC cells were cultured at 2x10 5 Cells / well were plated, and different concentrations of fusion protein were added, starting with 36 nM concentration, and diluted 4-fold to 8 concentration points. After incubation at 37°C in a cell culture incubator for 5 days, the supernatant was collected and used to detect the IFN-γ content using a human IFN-γ ELISA kit. The results are shown in Figure 9 and Table 8.

[0222] Table 8: Summary of CLDN18.2-Pro-IL-2 fusion protein promoting the release of IFN-γ cytokine by primary T cells

[0223] Experimental results showed that ICP-068+, ICP-106+, ICP-07+, and ICP-087+ all promoted concentration-dependent IFN-γ cytokine release and significantly enhanced IFN-γ release in PBMC-derived primary T cells compared to recombinant human IL-2. ICP-087- and ICP-106- exhibited slightly increased background activity in the uncleaved state compared to ICP-070- and ICP-106-.

[0224] IFN-γ mediates T cell killing of tumor cells through multiple pathways. The results showed that the inactivated form of CLDN18.2 fusion protein can effectively reduce T cell proliferation, activation and cytokine release when it is not cleaved by the MMP14 enzyme in the tumor microenvironment, and has the effect of reducing peripheral toxicity; at the same time, the active form of CLDN18.2 fusion protein after MMP14 cleavage can maintain the steady-state expansion of T cells, promote T cell activation and the release of cytokines such as IFN-γ. Its ability to release IFN-γ is stronger than that of recombinant IL-2, demonstrating its potent tumor-killing effect.

[0225] Example 8: Detection of ADCC activity of primary NK cells mediated by defucosylated CLDN18.2-Pro-IL-2 antibody fusion protein

[0226] In vivo, ADCC occurs when the Fab end of an antibody binds to an antigenic epitope on a tumor cell, and its Fc end binds to the FCγR on the surface of natural killer (NK) cells. Activated NK cells release cytotoxic substances such as perforin and granzymes, mediating NK cell killing of target cells and causing apoptosis. In this experiment, we used primary NK cells derived from peripheral blood mononuclear cells (PBMCs) as effector cells and 293T-hCLDN18.2 (cell number KC-0986, purchased from Kangyuan Broad) as target cells to establish a co-culture system to simulate the ADCC effect of antibodies in vitro.

[0227] First, target cells 293T-hCLDN18.2 were labeled with 1.6 μM CFSE (Carboxyfluorescein Diacetate Succinimidyl Ester, 565082, BD) for 10 minutes at room temperature in the dark. The cells were washed twice with 5 volumes of pre-cooled serum-free medium, resuspended in ADCC medium and counted. 5 x 10 cells were added to each well of a 96-well U-shaped plate. 4 Cells were then added with ICP-068, ICP-106, ICP-070 and ICP-087 active and inactive fusion proteins for gradient dilution. After centrifugation at 500 rpm for 30 seconds, 1.5 x 10 5PBMC cells / well, the effector-target ratio was 30:1. The target cell-effect cell-antibody fusion protein complex was incubated at 37°C for 4 hours. After incubation, the cells were washed twice with PBS + 2% FBS, and 100 μL of PBS solution containing 1 μL 7-AAD (559925, BD) was added to each well for staining. The cells were incubated in the dark at room temperature for 10 minutes, and the activity of the target cells was detected using a NovoCyte Quanteon flow cytometer (Agilent). The data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was fitted using nonlinear S-curve regression to obtain the data, and the EC was calculated from this. 50 The results are shown in Figures 10A, B and Table 9.

[0228] Table 9: ADCC activity of primary NK cells mediated by CLDN18.2-Pro-IL-2 antibody fusion protein EC 50 Summarize

[0229] The results showed that compared with the H7E12-2 mAb, ICP-087, due to Fc-terminal defucosylation, showed 1.69-fold and 4.33-fold increases in ADCC activity in the cleaved and uncleaved conditions, respectively (Figure 10A). Compared with the 432 mAb, the ADCC activity of ICP-106+ and ICP-106- increased by 2.76-fold and 1.65-fold, respectively (Figure 10B).

[0230] ADCC activity is an important immunomodulatory mechanism by which the CLDN18.2 antibody exerts its tumor-killing effect. The ADCC activity of the fucose-depleted CLDN18.2-Pro-IL-2 fusion protein is significantly improved compared to the Fc wild-type monoclonal antibody, indicating that the fusion protein structure does not affect the function of the Fc terminus of the CLDN18.2 antibody. Furthermore, the fusion protein produced by host cells with FUT8 or FUCT1 knockout in the present invention effectively enhances the ADCC activity of CLDN18.2-Pro-IL-2, enabling targeted killing of tumor cells with high CLDN18.2 expression, providing a theoretical basis for its clinical application.

[0231] In addition, the CLDN18.2-Pro-IL-2 fusion protein of the present invention, after enzymatic cleavage, releases IL-2, which can effectively activate the function of NK cells in the tumor microenvironment and further enhance the ADCC function of the CLDN18.2 antibody. Therefore, the fusion protein design of the present invention improves the ADCC activity of the CLDN18.2 antibody through two pathways, and has more significant tumor killing activity than the monoclonal antibody drugs currently in the clinical development stage.

[0232] Example 9: CLDN18.2-Pro-IL-2 Antibody Fusion Protein-Mediated Jurkat-NFAT-Luc2-CD16a-V158 ADCC Reporter Cell Line Activity Detection

[0233] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the main mechanism of action for many antibody drugs to kill tumor cells. FcγRⅢA (CD16a) can mediate the ADCC effect of NK cells on tumor cells. Here, Jurkat-NFAT-Luc2-CD16a-V158 (constructed by Kangyuan Bochuang) was used as an alternative effector cell and was co-incubated with target cells 293T-hCLDN18.2 (cells purchased from Kangyuan Bochuang, cell number KC-0986) overexpressing human CLDN18.2 as target cells at a 1:1 effector-target ratio. The extracellular region of this effector cell overexpressed high-affinity FcγRⅢA CD16a-V158 and was co-incubated with target cells and different concentrations of CLDN18.2-Pro-IL-2 fusion protein (86.9nM starting, 4-fold dilution, 9 concentration points). The Fc end of the fusion protein bound to the high-affinity extracellular region CD16a-V158 to activate the NFAT-luc2 luciferase reporter system. The ADCC activity of the ADCC-enhanced CLDN18.2-Pro-IL-2 fusion protein was determined using a luciferase assay (BMG Labtech). Data were analyzed using GraphPad Prism 7.0 software. Nonlinear sigmoid-curve regression was used to fit the data to generate dose-effect curves, from which EC50 values ​​were calculated. The experimental results are shown in Figure 11 and Table 10.

[0234] Table 10: Jurkat-NFAT-Luc2-CD16a-V158 ADCC reporter cell line EC mediated by CLDN18.2-Pro-IL-2 antibody fusion protein 50 Summarize

[0235] The results showed that among the CLDN18.2-Pro-IL-2 fusion proteins with H7E12-2 as the backbone, the ADCC activity EC values ​​of ICP-070 (Fc wild type), ICP-153 (ADCC enhanced type with amino acid mutation), ICP-087 (N297 defucosylated type, 292T-FUT8-), and ICP-155 (N297 defucosylated type, CHO-K1-AF) were significantly higher than those of ICP-070 (Fc wild type), ICP-153 (ADCC enhanced type with amino acid mutation), ICP-087 (N297 defucosylated type, 292T-FUT8-), and ICP-155 (N297 defucosylated type, CHO-K1-AF) 50The ADCC activity EC values ​​of ICP-068 (Fc wild type), ICP-154 (ADCC-enhanced type with S239D and I332E mutations), ICP-106 (N297 defucosylated type, 292T-FUT8-), and ICP-198 (N297 defucosylated type, CHO-K1-FUT8-) in the CLDN18.2-Pro-IL-2 fusion protein with 432 as the backbone were 0.98 nM, 0.02 nM, 0.04 nM, and 0.04 nM, respectively ( FIG11A ); the ... 50 0.54 nM, 0.02 nM, 0.04 nM and 0.05 nM, respectively ( FIG11B ).

[0236] Using antibody fusion protein mutants as templates, such as ICP-415, ADCC-enhancing mutations were performed to obtain ICP-501 (S239D and I332E mutations), ICP-502 (S239D, A330L and I332E mutations), and ICP-503 (F243L, R292P, Y300L, V305I, and P396L mutations). ADCC reporter cell line activity was detected using Jurkat-NFAT-Luc2-CD16a-V158 and Jurkat-NFAT-Luc2-CD16a-F158. The results are shown in Figures 11C and 11D and Table 11. The ADCC activity Ec50 of ICP-501, ICP-502 and ICP-503 in the CD16a-V158 subtype were 4.1pM, 4.1pM and 4.9pM, respectively. Compared with the unmutated ICP-415, the ADCC effect was increased by 11.0 times, 11.0 times and 9.2 times, respectively; while the ADCC activity Ec50 in the CD16a-V158 subtype was 15pM, 9.3pM and 25pM, respectively. Compared with the mutated ICP-415, the ADCC effect was increased by 16.5 times, 26.5 times and 9.9 times, respectively.

[0237] Table 11: Summary of the results of CLDN18.2-Pro-IL-2 antibody fusion protein-mediated ADCC in Jurkat-NFAT-Luc2-CD16a-V158 and Jurkat-NFAT-Luc2-CD16a-F158 reporter cell lines

[0238] Both different antibody ends and different fusion protein mutants showed that ADCC-enhancing mutations and fucose removal at position N297 effectively increased the ADCC activity of the CLDN18.2-Pro-IL-2 fusion protein by 13.5 to 27 times, thereby enhancing the cytotoxic activity against CLDN18.2-highly expressed tumor cells, providing a theoretical basis for its clinical application.

[0239] Example 10: Detection of STAT5 phosphorylation in NK-92 cells mediated by CLDN18.2-Pro-IL-2 antibody fusion protein

[0240] Active IL-2 activates the downstream JAK1 / JAK3-STAT5 signaling pathway by binding to IL-2Rβγ dimers on the surface of NK cells or CD8+ T cells, or IL-2Rαβγ trimers on the surface of Treg cells, promoting STAT5 phosphorylation (p-STAT5). Therefore, the ability of cleaved and uncleaved CLDN18.2-Pro-IL-2 fusion proteins to promote STAT5 phosphorylation in NK-92 cells was compared. 5 NK-92 cells (100 μL / mL) were resuspended in MEM basal medium and transferred to a 96-well cell culture plate, with 90 μL per well. The antibody was diluted three-fold in MEM basal medium starting at a concentration of 3 nM for a total of 10 concentration points. 10 μL of the antibody solution was transferred to the 96-well plate and incubated in a 37°C incubator for 15 minutes. NK-92 cells were washed twice with PBS buffer, then fixed with 100 μL of cold 90% methanol at 4°C for 30 minutes. They were then washed twice with PBS buffer, followed by the addition of Alexa Fluor 647-labeled antibody diluted according to the pSTAT5 antibody manufacturer's instructions. The cells were incubated at room temperature for half an hour, washed twice with PBS buffer, and STAT5 phosphorylation levels were measured using a NovoCyte Quanteon flow cytometer (Agilent). The results are shown in Figures 12A-D and Table 12.

[0241] Table 12: ECs of CLDN18.2-Pro-IL-2 antibody fusion protein promoting STAT5 phosphorylation in NK-92 cells 50 Summarize

[0242] The results showed that H7E12-2 and 432 monoclonal antibodies could not promote STAT5 phosphorylation, while enzyme-cleaved ICP-068+, ICP-106+, ICP-70+ and ICP-087+ promoted STAT5 phosphorylation in NK-92 cells. 50 The activity of recombinant IL-2 (EC 50 The inactive forms of ICP-068-, ICP-070-, ICP-087- and ICP-106- that were not digested had no ability to promote STAT5 phosphorylation ( Figures 12A-12D ).

[0243] Example 11: Efficacy of CLDN18.2-Pro-IL-2 Antibody Fusion Protein in MC-38-hCLDN18.2 and CT-26-hCLDN18.2 Mouse Colon Cancer Cell Models

[0244] The inventors simultaneously enrolled three groups of patients using the activatable antibody fusion protein Hit2.2-Pro-IL2 (protein number ICP-024), the fusion protein Fc-Pro-IL2 (protein number ICP-015), and the antibody form of Hit2.2-Pro-IL2 Hit2.1 (protein number ICP-025). They administered equimolar doses to perform efficacy tests in the MC-38-hCLDN18.2-A11 and CT-26-hCLDN18.2 mouse colon cancer cell models.

[0245] MC-38-hCLDN18.2-A11 mouse colon cancer cells (purchased from Nanjing Bowang) in the logarithmic growth phase were inoculated into 6-8 week old female C57BL / 6 mice (Weitong Lihua) at an inoculation volume of 5×10 5 / mouse, the inoculation volume was 0.1mL. When the tumor volume reached 60-80mm 3 At 14:00, intraperitoneal administration was started, and the administration cycle was BIW (twice a week). The drug was supplied 5 times, and the dosage was 0.26nmol / animal. After tumor inoculation, routine monitoring included tumor growth and the effect of treatment on the normal behavior of the animals. The specific contents included the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, hair and other abnormalities. The clinical symptoms observed during the experiment were recorded in the original data. The average tumor volume of the control group exceeded 2000mm 3 The experimental endpoint was set as the experimental end point, and spleen, draining lymph nodes and tumor tissues were collected for immune cell infiltration analysis.

[0246] Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b2)(where a represents the major diameter and b represents the minor diameter).

[0247] The relative tumor inhibition rate (TGI) (%) was calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the tumor weights (TW) of the treatment group and the PBS control group at a specific time point, respectively.) The tumor growth curve is shown in Figure 13.

[0248] In the MC-38-hCLDN18.2-A11 model, ICP-024 achieved a tumor inhibition rate (TGI) of 81.64% on day 23 (p<0.01, one-way ANOVA). This compares to the TGIs of the control drug ICP-015 and the control monoclonal antibody ICP-025, which were -35.06% and 32.21%, respectively, failing to reach significance compared to the PBS control group. Tumor growth curves for individual mice are shown in Figures 14A-D. Four of eight (50%) mice in the ICP-024-treated group achieved a complete response (CR) (Figure 14B), demonstrating the excellent anti-tumor activity of ICP-024 monotherapy in the MC-3-8 hCLDN18.2 mouse colon cancer model.

[0249] In the MC-38-hCLDN18.2-A11 model, the antibody fusion protein mutants ICP-414, ICP-415, and ICP-416 were administered, and the dose was reduced to 0.13 nmol / mouse. On day 23, tumors were measured. The tumor inhibition rate TGI of ICP-414 was 70.44% (p < 0.01, Oneway ANOVA), and 2 / 10 (20%) mice had complete tumor regression, achieving a complete response (Figure 15); the tumor inhibition rate TGI of ICP-415 was 83.77% (p < 0.01, Oneway ANOVA), and 7 / 10 (70%) mice had complete tumor regression, achieving a complete response (Figure 16); and the tumor inhibition rate TGI of ICP-416 was 100.01% (p < 0.01, Oneway ANOVA). ANOVA), and 8 / 10 (80%) mice had complete tumor regression, achieving a complete response ( Figure 17 ). Compared with the PBS control group, the efficacy of ICP-414, ICP-415, and ICP-416 were all significant, demonstrating that the antibody fusion protein mutants have significant anti-tumor ability in the MC38-hCLDN18.2 mouse colon cancer model.

[0250] The tumor inhibition rate of ICP-024 was also compared with that of the control test drugs ICP-015 and ICP-025 using the mouse colon cancer model CT-26-hCLDN18.2. CT-26-hCLDN18.2 mouse colon cancer cells (purchased from Nanjing Bowang) in the logarithmic growth phase were inoculated into 6-8 week old BALb / c female mice (Weitong Lihua) at a seeding volume of 5×10 5 / mouse, the inoculation volume is 0.1mL. When the tumor reaches 60-80mm 3Intraperitoneal administration was started at 5 pm, with a dosing cycle of BIW (twice a week) x 5 times, and a dose of 0.26 nmol / mouse. The experimental grouping, dosing, tumor measurement, and TGI calculation were the same as those for MC-38-hCLDN18.2-A11. The average tumor volume of the control group exceeded 2000 mm 3 The experimental endpoint was set as the experimental endpoint, and peripheral blood was collected for absolute immune cell count analysis to detect its potential peripheral toxicity. The tumor growth curve is shown in Figure 18. In the CT-26-hCLDN18.2 model, on day 16, the tumor inhibition rate TGI of ICP-024 was 97.99% (p<0.05, Oneway ANOVA), while the TGI of the control drug ICP-015 and the control monoclonal antibody ICP-025 were 56.12% and -33.81%, respectively, which were not significant compared with the PBS control group; the tumor growth curves of individual mice are shown in Figures 19A-D. In the ICP-024 administration group, 5 / 8 (62.5%) mice achieved complete tumor clearance (CR, complete response), and 2 / 8 (25%) mice had tumors less than 70mm 3 Only one mouse (12.5%) developed tumor progression (PD), demonstrating the excellent anti-tumor ability of ICP-024 monotherapy in the CT-26-hCLDN18.2 mouse colon cancer model.

[0251] Peripheral blood immune cell count analysis, as shown in Figures 20A-D, examined potential peripheral immune toxicity in each treatment group at the efficacy endpoint after repeated dosing. The absolute peripheral blood immune cell counts demonstrated that ICP-015 promoted the expansion of peripheral CD45+ immune cells, CD3+ T cells, and CD8+ T cells compared to the control group, while ICP-024 demonstrated good peripheral safety and did not cause excessive activation or expansion of peripheral blood immune cells.

[0252] These results suggest that the antibody-targeted CLDN18.2-Pro-IL-2 can specifically target the tumor site and reduce peripheral nonspecific T and NK cell activation. When the fusion protein is targeted to the tumor site, it can effectively activate T cells and NK cells in the tumor microenvironment, promote tumor killing, and simultaneously exert an anti-tumor effect of reducing toxicity and increasing efficacy.

[0253] Example 12: CLDN18.2-Pro-IL-2 antibody fusion protein induces immune memory formation in the MC-38-hCLDN18.2 mouse colon cancer cell model

[0254] Furthermore, the CLDN18.2-Pro-IL-2 fusion protein (ICP-069) with the reference antibody Zolbetuximab as the backbone was compared with the fusion proteins composed of several other CLDN18.2 antibodies, namely ICP-024, ICP-068, ICP-069, ICP-070 and ICP-087, in the MC-38-hCLDN18.2-A11 mouse colon cancer tumor model for tumor suppression and the ability to promote immune memory formation. The MC-38-hCLDN18.2-A11 tumor modeling and grouping, and tumor size detection were the same as in Example 11 above. When the tumor size reached 60-80 mm 3 Intraperitoneal administration was initiated at 4:00 AM, with a dosing cycle of BIW (twice weekly) for a total of five times, at a dose of 260 nmol / mouse. As shown in Figure 21 , on day 14 of tumor growth, compared to the control group, the tumor inhibition rate (TGI) for ICP-024 was 93.75% (p < 0.0001, one-way ANOVA), the tumor inhibition rate (TGI) for ICP-068 was 100.94% (p < 0.0001, one-way ANOVA), the tumor inhibition rate (TGI) for ICP-069 was 67.38% (p < 0.001, one-way ANOVA), the tumor inhibition rate (TGI) for ICP-070 was 59.58% (p < 0.01, one-way ANOVA), and the tumor inhibition rate (TGI) for ICP-087 was 108.84% (p < 0.0001, one-way ANOVA). Tumor growth curves for individual mice are shown in Figures 22A-F. At the endpoint of the experiment, 7 / 8 (87.5%) mice in the ICP-024 group achieved complete tumor regression, 3 / 8 (37.5%) mice in the ICP-068 group achieved complete tumor regression, 2 / 8 (25%) mice in the ICP-069 group achieved complete tumor regression, no mice in the ICP-070 group achieved complete tumor regression, and 8 / 8 (100%) mice in the ICP-087 group achieved complete tumor regression.

[0255] Mice with complete tumor regression (from ICP-024 and ICP-087 groups) continued routine feeding and tumor monitoring for 6 weeks, after which they were re-inoculated on the contralateral side with 5 x 10 5 MC-38-hCLDN18.2-A11 (purchased from Nanjing Bowang) cells / mouse, and 8 wild-type mice (Weitong Lihua) were inoculated as tumor control. Tumor growth was measured weekly. The average tumor size of the control group mice was about 1200mm 3Observation was terminated at 14:00. As shown in Figure 23, compared with the control group, no contralateral tumors progressed in the ICP-024 group, while one mouse in the ICP-087 group developed tumor progression. The contralateral tumor growth curves of individual mice are shown in Figures 24A-C. The tumor incidence rate of the contralateral inoculation of ICP-024 was 0% (0 / 70); the contralateral tumor incidence rate of ICP-087 was 12.5% ​​(1 / 8), and only one mouse developed contralateral tumor growth. These results show that ICP-024 and ICP-087 generate anti-tumor-specific immune memory in the process of promoting tumor killing. When the same tumor antigen is encountered again, the immune memory response can be effectively activated and promote tumor killing.

[0256] Example 13: Toxicity Study of CLDN18.2-Pro-IL-2 Antibody Fusion Protein in MC-38-hCLDN18.2 Mouse Colon Cancer Cell Model

[0257] The main toxicity of IL-2 is capillary leakage and multiple organ failure. We used MC-38-hCLDN18.2 tumor-bearing mice to study the toxicity of CLDN18.2-Pro-IL-2. Mouse tumor cell inoculation and tumor measurement were as described in Example 12. When tumors developed and reached 500-800 mm 3 A single intraperitoneal administration of 0.26 nmol / mouse was performed. 96 hours after administration, the lungs of the mice were harvested and weighed, dried at 37°C for 48 hours, and weighed again. The difference was the net weight.

[0258] The results are shown in Figure 25. 96 hours after a single equimolar dose of Fc-IL-2, there was a significant increase in lung net weight compared to the control group, indicating capillary leakage, while the same dose of ICP-106 did not increase lung net weight compared to the control, indicating that the CLDN18.2-Pro-IL-2 antibody fusion protein has good peripheral safety.

[0259] Example 14: Preparation and Activity Verification of Trastuzumab-Pro-IL-2 Antibody Fusion Protein

[0260] The connection method of Trastuzumab-Pro-IL-2 to IL2 and its shielding peptide is as described in Example 1. The heavy chain and light chain sequences are shown in SEQ ID NO: 42 and SEQ ID NO: 41, and it is named ICP-269.

[0261] Its expression was as described in Example 2. The CHO-S cell density was adjusted to 6×10 6 / mL, liposomes were used for transfection, the plasmid concentration was 1mg / ml, and cultured at 32°C, 5% CO2, 100rpm for 7-10 days. Feeds were added 18-22h after transfection and between the 5th day. The supernatant was collected by ultrafiltration, and the protein was purified by ProteinA, ion exchange column or molecular sieve. The collection tube where the target protein was located was selected according to UV280 absorption and stored together. The purified protein was ultrafiltered and exchanged into the target buffer. The protein purity and content were detected by SDS-PAGE gel electrophoresis and SEC-HPLC high-performance liquid chromatography (Figure 26A and Figure 27A). The results showed that ICP-269 had a high purity after one-step purification by protein A, with a purity greater than 95%.

[0262] The binding ability of ICP-269 was tested using BT474 cells, which highly express Her2. The binding constant Ec50 was 4.8 nM, which is comparable to the trastuzumab binding constant Ec50 of 3.9 nM ( Figure 29A , Table 13 ). This result indicates that the binding ability of the antibody end of trastuzumab is not affected when it is formulated as an antibody fusion protein.

[0263] Table 13: Binding constants of ICP-269 and Her2-overexpressing cells BT474

[0264] ICP-269 was digested with MMP14, and the digested protein was electrophoresed under non-reducing and reducing conditions, as shown in Figures 28A and 28B. The digested protein was tested for IL-2 activity in CTLL-2 and NK-92 cell proliferation assays (Figures 30A and 30B). The results showed that the fusion protein ICP-269+, i.e., ICP-269 after digestion, had a high activity in EC of NK-92 cells. 50 were 7.15±3.32nM, respectively, which is comparable to the EC values ​​of human recombinant IL-2. 50 activity 10.45 ± 0.78 equivalent to uncleaved ICP-269-, whose EC in NK-92 cells 50 Compared with 761.5±94.05nM, the activity was reduced by 116.5±40.31 times (Figure 30A, Table 15). 50 The fold change was 195±36.77 and 14±4.24, respectively, with a fold change of 14±1.41. The results showed that the inactive form of the trastuzumab-Pro-IL-2 fusion protein significantly reduced cytokine IL-2 activity. However, after MMP14 cleavage, IL-2 was effectively released, promoting NK cell expansion. Trastuzumab-Pro-IL-2 exhibits the characteristics of an activatable antibody fusion protein (Figure 30B, Table 16).

[0265] Example 15: Protein Preparation and Activity Verification of Rituximab-Pro-IL-2 Antibody Fusion Protein

[0266] The linkage of Rituximab-Pro-IL-2 to IL-2 and its shielding peptide was as described in Example 1. Its sequence is shown in SEQ ID NO:44 and SEQ ID NO:43, and it was designated ICP-270. Its expression, purification, and characterization were as described in Example 13. Purification results were determined by SDS-PAGE gel electrophoresis and SEC-HPLC high-performance liquid chromatography. Protein purity and content are shown in Figures 26B and 27B. The results demonstrate that ICP-270 is highly pure after one-step purification with protein A, exceeding 95%.

[0267] ICP-270 binding was assessed using REC-1 cells, which highly express CD20. The binding constant, Ec50, was 7.0 nM, comparable to the rituximab binding constant of 2.4 nM (Figure 29B, Table 14). This result demonstrates that the binding capacity of rituximab, when formulated as an antibody fusion protein, is not affected. Table 14 shows the binding constants for ICP-270 and REC-1 cells, which highly express CD20.

[0268] Table 14: Binding constants of ICP-270 and CD20-high expressing REC-1 cells

[0269] ICP-270 was cleaved by MMP14, and the cleaved proteins were run on non-reducing and reducing gels, as shown in Figures 28A and 28B. The cleaved proteins were then tested for IL-2 activity using CTLL-2 and NK-92 cell proliferation assays (Figures 30A and 30B). The results showed that the EC50 of the fusion protein ICP-270+ (i.e., the cleaved ICP-270) in NK-92 cells was 9.15±1.2 nM, comparable to the EC50 activity of human recombinant IL-2 of 10.45±0.78. The activity of the uncleaved ICP-270- was 408±142.84 nM in NK-92 cells, representing a 45.5±21.92-fold decrease. The EC50 values ​​of the fusion protein ICP-270 for the inactive and active forms of CTLL-2 were 208±15.56 and 17±2.83, respectively, representing a fold change of 12.5±3.54. The results showed that the cytokine IL-2 activity of the inactive Rituximab-Pro-IL-2 fusion protein was significantly reduced. However, after MMP14 cleavage, IL-2 was effectively released, promoting NK cell expansion. Rituximab-Pro-IL-2 exhibits the characteristics of an activatable antibody fusion protein.

[0270] Table 15: Summary of EC50 and fold change of ICP-269 and ICP-270 on NK-92 cell proliferation

[0271] Table 16: Summary of EC50 and fold change of ICP-269 and ICP-270 on CTLL-2 cell proliferation

[0272] In summary, in vitro and in vivo activity experiments showed that the CLDN18.2-Pro-IL-2, Trastuzumab-Pro-IL-2, and Rituximab-Pro-IL-2 fusion proteins, when not cleaved by MMP14, effectively inhibited the activity of the cytokine IL-2 (including promoting the phosphorylation of STAT5 in T cells or NK cells, thereby promoting the expansion of NK and T cells, and the release of the cytokine IFN-γ). This indicates that the inactivated antibody fusion protein provided by the present invention blocks the binding of IL-2 to the receptor IL-2Rβγ on T cells or NK cells by shielding the peptide IL-2Rα, thereby not activating immune cells in the peripheral blood, thereby achieving the purpose of reducing peripheral systemic immunotoxicity. In addition, after cleavage by MMP14 in the tumor microenvironment, the activated antibody fusion protein releases active IL-2 through the cleavable connecting peptide, thereby specifically increasing the phosphorylation of STAT5 in T cells or NK cells in the tumor microenvironment, promoting the expansion of NK and T cells, and the release of the cytokine IFN-γ, thereby achieving the effect of tumor killing.

[0273] On the other hand, the CLDN18.2-Pro-IL-2, Trastuzumab-Pro-IL-2 and Rituximab-Pro-IL-2 fusion proteins provided by the present invention exert a direct tumor-killing effect. The ADCC-enhanced CLDN18.2-Pro-IL-2 fusion protein provided by the present invention effectively improves the ADCC activity of the fusion protein, further improves its ADCC activity, and thereby produces antibody-mediated cell killing of CLDN18.2-positive tumor cells.

[0274] In a third aspect, the CLDN18.2-Pro-IL-2, Trastuzumab-Pro-IL-2 and Rituximab-Pro-IL-2 fusion proteins provided by the present invention, with an ADCC-enhanced Fc end and an enzymatically cleaved and activated IL-2 cytokine end, further increase the function of the antibody fusion protein by jointly acting on NK cells locally infiltrating the tumor microenvironment, thereby achieving a synergistic effect.

[0275] Therefore, the present invention provides a generally applicable antibody fusion protein form, which can promote the killing of target cells by immune cells through multiple molecular mechanisms, while effectively reducing the peripheral toxicity of IL-2 cytokines. The antibody fusion protein drug provided by the present invention has good tumor treatment effect and safety, and is a highly effective and low-toxic antibody cytokine fusion protein product.

[0276] References

[0277] Atkins,MB,Lotze,MT,Dutcher,JP,Fisher,RI,Weiss,G.,Margolin,K.,Rosenberg,SA(1999).High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma:analysis of 270 patients treated between 1985 and 1993.J Clin Oncol,17(7),2105-2116.doi:10.1200 / jco.1999.17.7.2105;

[0278] Charych,D.,Khalili,S.,Dixit,V.,Kirk,P.,Chang,T.,Langowski,J.,Zalevsky,J.(2017).Modeling the receptor pharmacology,pharmacokinetics,and pharmacodynamics of NKTR-214,a kinetically-controlled interleukin-2(IL2)receptor agonist for cancer immunotherapy.PLoS One,12(7),e0179431.doi:10.1371 / journal.pone.0179431;

[0279] Charych,D.H.,Hoch,U.,Langowski,J.L.,Lee,S.R.,Addepalli,M.K.,Kirk,P.B.,Doberstein,S.K.(2016).NKTR-214,an Engineered Cytokine with Biased IL2 Receptor Binding,Increased Tumor Exposure,and Marked Efficacy in Mouse Tumor Models.Clin Cancer Res,22(3),680-690.doi:10.1158 / 1078-0432.CCR-15-1631;

[0280] Gribskov,M.R.,&Devereux,J.(1991).Sequence analysis primer.New York Basingstroke,Hants,England:Stockton Press Macmillan Publishers;

[0281] Griffin,A.M.,&Griffin,H.G.(1994).Computer analysis of sequence data.Totowa,N.J.:Humana Press;

[0282] Heijne,G.v.(1987).Sequence analysis in molecular biology:treasure trove or trivial pursuit.San Diego:Academic Press;

[0283] Hernandez,R.,Poder,J.,LaPorte,K.M.,&Malek,T.R.(2022).Engineering IL-2 for immunotherapy of autoimmunity and cancer.Nat Rev Immunol.doi:10.1038 / s41577-022-00680-w

[0284] Hsu,E.J.,Cao,X.,Moon,B.,Bae,J.,Sun,Z.,Liu,Z.,&Fu,Y.X.(2021).A cytokine receptor-masked IL2 prodrug selectively activates tumor-infiltrating lymphocytes for potent antitumor therapy.Nat Commun,12(1),2768.doi:10.1038 / s41467-021-22980-w;

[0285] Jovov,B.,Van Itallie,C.M.,Shaheen,N.J.,Carson,J.L.,Gambling,T.M.,Anderson,J.M.,&Orlando,R.C.(2007).Claudin-18:adominant tight junction protein in Barrett's esophagus and likely contributor to its acid resistance.Am J Physiol Gastrointest Liver Physiol,293(6),G1106-1113.doi:10.1152 / ajpgi.00158.2007;

[0286] Karanjawala,Z.E.,Illei,P.B.,Ashfaq,R.,Infante,J.R.,Murphy,K.,Pandey,A.,Hruban,R.H.(2008).New markers of pancreatic cancer identified through differential gene expression analyses:claudin 18 and annexin A8.Am J Surg Pathol,32(2),188-196.doi:10.1097 / PAS.0b013e31815701f3

[0287] Klamp,T.,Schumacher,J.,Huber,G.,Kuhne,C.,Meissner,U.,Selmi,A.,Sahin,U.(2011).Highly specific auto-antibodies against claudin-18 isoform 2 induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases.Cancer Res,71(2),516-527.doi:10.1158 / 0008-5472.CAN-10-2292;

[0288] Krause,G.,Winkler,L.,Mueller,S.L.,Haseloff,R.F.,Piontek,J.,&Blasig,I.E.(2008).Structure and function of claudins.Biochimica et Biophysica Acta(BBA)-Biomembranes,1778(3),631-645.doi:https: / / doi.org / 10.1016 / j.bbamem.2007.10.018;

[0289] Pires,I.S.,Hammond,P.T.,&Irvine,D.J.(2021).Engineering Strategies for Immunomodulatory Cytokine Therapies-Challenges and Clinical Progress.Adv Ther(Weinh),4(8).doi:10.1002 / adtp.202100035;

[0290] Sahin,U.,Tureci,O.,Manikhas,G.,Lordick,F.,Rusyn,A.,Vynnychenko,I.,Al-Batran,S.E.(2021).FAST:a randomised phase II study of zolbetuximab(IMAB362)plus EOX versus EOX alone for first-line treatment of advanced CLDN18.2-positive gastric and gastro-oesophageal adenocarcinoma.Ann Oncol,32(5),609-619.doi:10.1016 / j.annonc.2021.02.005;

[0291] Schwartz,R.N.,Stover,L.,&Dutcher,J.P.(2002).Managing toxicities of high-dose interleukin-2.Oncology(Williston Park),16(11 Suppl 13),11-20;

[0292] Smith,D.W.(1994).Biocomputing:informatics and genome projects.San Diego:Academic Press.

[0293] Welschof,M.,&Krauss,J.r.(2003).Recombinant antibodies for cancer therapy:methods and protocols.Totowa,N.J.:Humana Press;

[0294] Xue,D.,Hsu,E.,Fu,Y.X.,&Peng,H.(2021).Next-generation cytokines for cancer immunotherapy.Antib Ther,4(2),123-133.doi:10.1093 / abt / tbab014。

Claims

1. An activatable antibody fusion protein, characterized in that It comprises an antibody portion that specifically binds to a target, an immunoglobulin Fc portion, a shielding portion, and a cytokine portion, wherein the shielding portion is fused to the immunoglobulin Fc portion via a connecting peptide L1, and the cytokine portion is fused to the shielding portion via a cleavable connecting peptide L2, wherein: a) the target is a tumor-specific antigen, wherein the tumor-specific antigen is selected from one or more of the following groups: Claudin18.2, CA125, AFP, CEA, EGFR, HER2, B7H3, B7H6, MUC1, MUC16, GPC3, CD24, CD20; The cytokine is selected from one or more of the following: interleukin-2 (IL-2), interferon alpha (IFNα), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin-7 (IL-7), interleukin-12 (IL-12), and interleukin-21 (IL-21); and / or The shielding portion is a receptor of the cytokine or a binding fragment thereof, or an antibody or a binding fragment thereof that specifically binds to the cytokine, and can inhibit the activity of the cytokine by binding to the cytokine.

2. The activatable antibody fusion protein according to claim 1, wherein: a) the tumor-specific antigen is selected from CLDN18.2, HER2 or CD20; preferably, the tumor-specific antigen is CLDN18.2; b) the cytokine is IL-2; preferably, the cytokine is wild-type IL-2, a mutant or a truncated variant thereof; and / or c) The shielding portion inhibits the activity of the cytokine by inhibiting the binding of IL-2 to IL-2Rαβγ and / or IL-2Rβγ on immune cells; preferably, the shielding portion is selected from: IL-2Rα, IL-2Rβ, IL-2Rγ or its mutants or truncated variants.

3. The activatable antibody fusion protein according to claim 1, wherein: a) wherein the connecting peptide L1 is selected from a flexible connecting peptide comprising glycine (G) and serine (S) residues; preferably, the connecting peptide L1 comprises (GGGGS) n Repeatedly, wherein n is selected from an integer of 1-6, and / or b) The cleavable connecting peptide L2 is cleaved by a tumor-associated protease, thereby releasing the active cytokine, wherein the protease is selected from matrix metallopeptidase-1 (MMP1), MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP19, MMP20, MMP21, uPA, FAPa or cathepsin B; or the protease is selected from caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase-cleaving 11 of caspase and caspase 12; preferably, the cleavable connecting peptide L2 is cleaved by matrix metallopeptidase 14.

4. The activatable antibody fusion protein according to claim 1, wherein: a) the amino acid sequence of the cytokine portion is shown in SEQ ID NO: 27, SEQ ID NO: 74 or SEQ ID NO: 86; b) the shielding moiety is IL-2Rα; preferably, the amino acid sequence of the shielding moiety is shown in SEQ ID NO: 29, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 85; c) the amino acid sequence of the connecting peptide L1 is shown in SEQ ID NO: 23; and / or d) The amino acid sequence of the connecting peptide L2 is shown in SEQ ID NO: 25, SEQ ID NO: 71, SEQ ID NO: 72 or SEQ ID NO:

73.

5. The activatable antibody fusion protein according to claim 1, wherein: a) the immunoglobulin Fc portion is selected from the constant region amino acid sequence of IgG1, IgG2, IgG3, and IgG4, preferably selected from the constant region amino acid sequence of IgG1, the amino acid sequence of which is shown in SEQ ID NO: 39; and / or b) the immunoglobulin Fc part comprises one or more amino acid substitutions selected from the group consisting of S239D, S298A, I332E and A330L, preferably S239D and I332E or S239D, I332E and A330L.

6. The activatable antibody fusion protein according to claim 1, wherein: a) the antibody portion that specifically binds to a target is selected from the group consisting of Fab, Fab', F(ab')2, Fv, dsFv, diabody, Fd, and Fd' fragments; or b) the antibody portion that specifically binds to the target forms an antibody structure comprising a heavy chain and a light chain with the immunoglobulin Fc portion, wherein: i) the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 3, 7, 11, 15, 42, and 44; and / or the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 9, 13, 17, 19, 21, 31, 37, 35, 33, 49, 51, 53, 55, 57, 59, 61, 63, 69, 75, 77, 79; or ii) the amino acid sequence of the light chain is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 5; or The amino acid sequence of the light chain is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 9; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 21; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 37; or The amino acid sequence of the light chain is shown in SEQ ID NO: 11; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13; or The amino acid sequence of the light chain is shown in SEQ ID NO: 11; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 35; or The amino acid sequence of the light chain is shown in SEQ ID NO: 15; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 17; or The amino acid sequence of the light chain is shown in SEQ ID NO: 15; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; or The amino acid sequence of the light chain is shown in SEQ ID NO: 15; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 33; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 49; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 51; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 53; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 55; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 57; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 59; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 61; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 63; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 69; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 75; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 77; or The amino acid sequence of the light chain is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain is shown in SEQ ID NO:

79.

7. The activatable antibody fusion protein of claim 1, wherein the fusion protein comprises, from N-terminus to C-terminus: An antibody portion that specifically binds to a target, such as the immunoglobulin Fc portion as shown in SEQ ID NO:39, a connecting peptide L1 as shown in SEQ ID NO:23, a shielding portion as shown in SEQ ID NO:29, a connecting peptide L2 as shown in SEQ ID NO:25, and a cytokine portion as shown in SEQ ID NO:27; preferably, the fusion protein comprises, from N-terminus to C-terminus: an antibody portion that specifically binds to a target and a sequence selected from either SEQ ID NO:1 or 87.

8. The activatable antibody fusion protein of claim 1, wherein the fusion protein is selected from the antibody fusion proteins listed in Table 1.

9. An isolated nucleic acid molecule comprising a polynucleotide encoding the activatable antibody fusion protein according to any one of claims 1 to 8. A host cell comprising the nucleic acid molecule of claim 9 .

11. The host cell according to claim 10, which has an altered glycosylation machinery such that fucose residues are not attached to sugar chains or such attachment is minimized, preferably the host cell lacks effective fucosyltransferase activity or fucose transport activity; preferably, the fucosyltransferase is FUT8 and / or the fucose transporter is FUCT1.

12. A method for producing the activatable antibody fusion protein according to any one of claims 1 to 8, comprising culturing the host cell according to any one of claims 10 or 11 to express the fusion protein, and isolating the expressed fusion protein.

13. The activatable antibody fusion protein product produced by the method according to claim 12, characterized in that The fucosylation level of Asn 297 at position Fc region of the immunoglobulin is reduced, preferably, the activatable antibody fusion protein with fucosylation modification at Asn 297 at position Fc region of the immunoglobulin accounts for 10% or less of the total amount of all activatable antibody fusion proteins; wherein the activatable antibody fusion protein without fucosylation has enhanced antibody-dependent cellular cytotoxicity compared to the fucosylated control fusion protein.

14. Use of the activatable antibody fusion protein according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, or the activatable antibody fusion protein product according to claim 13 in the preparation of a drug or reagent for diagnosing, treating, or preventing tumors or autoimmune diseases.

15. The use according to claim 14, characterized in that The tumor is a tumor associated with CLDN18.2, a tumor associated with HER2, or a tumor associated with CD20; preferably, the tumor is gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, breast cancer, lymphoma or leukemia; the autoimmune disease is selected from rheumatoid arthritis, autoimmune hemolytic anemia, pure red cell aplasia, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, Evans syndrome, vasculitis, and bullous skin disease.

Images